Mass spectrometric methods for biomolecular screening

ABSTRACT

The present invention provides methods for the determination of the structure of biomolecular targets, as well as the site and nature of the interaction between ligands and biomolecular targets. The present invention also provides methods for the determination of the relative affinity of a ligand for the biomolecular target it interacts with. Also provided are methods for screening ligand or combinatorial libraries of compounds against one or more than one biological target molecules. The methods of the invention also allow determination of the relative binding affinity of combinatorial and other compounds for a biomolecular target. The present invention further provides methods for the use of mass modifying tags for screening multiple biomolecular targets. In a preferred embodiment, ligands which have great specificity and affinity for molecular interaction sites on biomolecules, especially RNA can be identified. In preferred embodiments, such identification can be made simultaneously with libraries of ligands.

BACKGROUND OF THE INVENTION

[0001] The process of drug discovery is changing at a fast pace becauseof the rapid progress and evolution of a number of technologies thatimpact this process. Drug discovery has evolved from what was, severaldecades ago, essentially random screening of natural products, into ascientific process that not only includes the rational and combinatorialdesign of large numbers of synthetic molecules as potential bioactiveagents, such as ligands, agonists, antagonists, and inhibitors, but alsothe identification, and mechanistic and structural characterization oftheir biological targets, which may be polypeptides, proteins, ornucleic acids. These key areas of drug design and structural biology areof tremendous importance to the understanding and treatment of disease.However, significant hurdles need to be overcome when trying to identifyor develop high affinity ligands for a particular biological target.These include the difficulty surrounding the task of elucidating thestructure of targets and targets to which other molecules may be boundor associated, the large numbers of compounds that need to be screenedin order to generate new leads or to optimize existing leads, the needto dissect structural similarities and dissimilarities between theselarge numbers of compounds, correlating structural features to activityand binding affinity, and the fact that small structural changes canlead to large effects on biological activities of compounds.

[0002] Traditionally, drug discovery and optimization have involved theexpensive and time-consuming, and therefore slow, process of synthesisand evaluation of single compounds bearing incremental structuralchanges. When using natural products, the individual components ofextracts had to be painstakingly separated into pure constituentcompounds prior to biological evaluation. Further, all compounds had tobe carefully analyzed and characterized prior to in vitro screening.These screens typically included evaluation of candidate compounds forbinding affinity to their target, competition for the ligand bindingsite, or efficacy at the target as determined via inhibition, cellproliferation, activation or antagonism end points. Considering allthese facets of drug design and screening that slow the process of drugdiscovery, a number of approaches to alleviate or remedy these matters,have been implemented by those involved in discovery efforts.

[0003] One way in which the drug discovery process is being acceleratedis by the generation of large collections, libraries, or arrays ofcompounds. The strategy of discovery has moved from selection of drugleads from among compounds that are individually synthesized and testedto the screening of large collections of compounds. These collectionsmay be from natural sources (Stemberg et al., Proc. Natl. Acad. Sci.USA, 1995, 92, 1609-1613) or generated by synthetic methods such ascombinatorial chemistry (Ecker and Crooke, Bio/Technology, 1995, 13,351-360 and U.S. Patent 5,571,902, incorporated herein by reference).These collections of compounds may be generated as libraries ofindividual, well-characterized compounds synthesized, e.g. via highthroughput, parallel synthesis or as a mixture or a pool of up toseveral hundred or even several thousand molecules synthesized bysplit-mix or other combinatorial methods. Screening of suchcombinatorial libraries has usually involved a binding assay todetermine the extent of ligand-receptor interaction (Chu et al., J. Am.Chem. Soc., 1996, 118, 7827-35). Often the ligand or the target receptoris immobilized onto a surface such as a polymer bead or plate. Followingdetection of a binding event, the ligand is released and identified.However, solid phase screening assays can be rendered difficult bynon-specific interactions.

[0004] Whether screening of combinatorial libraries is performed viasolid-phase, solution methods or otherwise, it can be a challenge toidentify those components of the library that bind to the target in arapid and effective manner and which, hence, are of greatest interest.This is a process that needs to be improved to achieve ease andeffectiveness in combinatorial and other drug discovery processes.Several approaches to facilitating the understanding of the structure ofbiopolymeric and other therapeutic targets have also been developed soas to accelerate the process of drug discovery and development. Theseinclude the sequencing of proteins and nucleic acids (Smith, in ProteinSequencing Protocols, Humana Press, Totowa, N.J., 1997; Findlay andGeisow, in Protein Sequencing: A Practical Approach, IRL Press, Oxford,1989; Brown, in DNA Sequencing, IRL Oxford University Press, Oxford,1994; Adams, Fields and Venter, in Automated DNA Sequencing andAnalysis, Academic Press, San Diego, 1994). These also includeelucidating the secondary and tertiary structures of such biopolymersvia NMR (Jefson, Ann. Rep. in Med. Chem., 1988, 23,275; Erikson andFesik, 30 Ann. Rep. in Med. Chem., 1992, 27, 271-289), X-raycrystallography (Erikson and Fesik, Ann. Rep. in Med.Chem.,1992,27,271-289) and the use of computer algorithms to attempt theprediction of protein folding (Copeland, in Methods of Protein Analysis:A Practical Guide to Laboratory Protocols, Chapman and Hall, New York,1994; Creighton, in Protein Folding, W. H. Freeman and Co., 1992).Experiments such as ELISA (Kemeny and Challacombe, in ELISA and otherSolid Phase Immunoassays: Theoretical and Practical Aspects; Wiley, NewYork, 1988) and radioligand binding assays (Berson and Yalow, Clin.Chim. Acta, 1968, 22, 51-60; Chard, in “An Introduction toRadioimmunoassay and Related Techniques,” Elsevier press, Amsterdam/NewYork, 1982), the use of surface-plasmon resonance (Karlsson, Michaelssonand Mattson, J. Immunol. Methods, 1991, 145, 229; Jonsson et al.,Biotechniques, 1991, 11, 620), and scintillation proximity assays(Udenfriend, Gerber and Nelson, Anal. Biochem., 1987, 161, 494-500) arebeing used to understand the nature of the receptor-ligand interaction.

[0005] All of the foregoing paradigms and techniques are now availableto persons of ordinary skill in the art and their understanding andmastery is assumed herein.

[0006] Likewise, advances have occurred in the chemical synthesis ofcompounds for high-throughput biological screening. Combinatorialchemistry, computational chemistry, and the synthesis of largecollections of mixtures of compounds or of individual compounds have allfacilitated the rapid synthesis of large numbers of compounds for invitro screening. Despite these advances, the process of drug discoveryand optimization entails a sequence of difficult steps. This process canalso be an expensive one because of the costs involved at each stage andthe need to screen large numbers of individual compounds. Moreover, thestructural features of target receptors can be elusive.

[0007] One step in the identification of bioactive compounds involvesthe determination of binding affinity of test compounds for a desiredbiopolymeric or other receptor, such as a specific protein or nucleicacid or combination thereof. For combinatorial chemistry, with itsability to synthesize, or isolate from natural sources, large numbers ofcompounds for in vitro biological screening, this challenge ismagnified. Since combinatorial chemistry generates large numbers ofcompounds or natural products, often isolated as mixtures, there is aneed for methods which allow rapid determination of those members of thelibrary or mixture that are most active or which bind with the highestaffinity to a receptor target.

[0008] From a related perspective, there are available to the drugdiscovery scientist a number of tools and techniques for the structuralelucidation of biologically interesting targets, for the determinationof the strength and stoichiometry of target-ligand interactions, and forthe determination of active components of combinatorial mixtures.

[0009] Techniques and instrumentation are available for the sequencingof biological targets such as proteins and nucleic acids (e.g. Smith, inProtein Sequencing Protocols, 1997 and Findlay and Geisow, in ProteinSequencing: A Practical Approach, 1989) cited previously. While thesetechniques are useful, there are some classes and structures ofbiopolymeric target that are not susceptible to such sequencing efforts,and, in any event, greater convenience and economy have been sought.Another drawback of present sequencing techniques is their inability toreveal anything more than the primary structure, or sequence, of thetarget.

[0010] While X-ray crystallography is a very powerful technique that canallow for the determination of some secondary and tertiary structure ofbiopolymeric targets (Erikson and Fesik, Ann. Rep. in Med. Chem., 1992,27, 271-289), this technique can be an expensive procedure and verydifficult to accomplish. Crystallization of biopolymers is extremelychallenging, difficult to perform at adequate resolution, and is oftenconsidered to be as much an art as a science. Further confounding theutility of X-ray crystal structures in the drug discovery process is theinability of crystallography to reveal insights into the solution-phase,and therefore the biologically relevant, structures of the targets ofinterest.

[0011] Some analysis of the nature and strength of interaction between aligand (agonist, antagonist, or inhibitor) and its target can beperformed by ELISA (Kemeny and Challacombe, in ELISA and other SolidPhase Immunoassays: 1988), radioligand binding assays (Berson and Yalow,Clin. 1968, Chard, in “An Introduction to Radioimmunoassay and RelatedTechniques,” 1982), surface-plasmon resonance (Karisson, Michaelsson andMattson, 1991, Jonsson et al., Biotechniques, 1991), or scintillationproximity assays (Udenfriend, Gerber and Nelson, Anal. Biochem., 1987),all cited previously. The radioligand binding assays are typicallyuseful only when assessing the competitive binding ofthe unknown at thebiding site for that of the radioligand and also require the use ofradioactivity. The surface-plasmon resonance technique is morestraightforward to use, but is also quite costly. Conventionalbiochemical assays of binding kinetics, and dissociation and associationconstants are also helpful in elucidating the nature of thetarget-ligand interactions.

[0012] When screening combinatorial mixtures of compounds, the drugdiscovery scientist will conventionally identify an active pool,deconvolute it into its individual members via resynthesis, and identifythe active members via analysis of the discrete compounds. Currenttechniques and protocols for the study of combinatorial librariesagainst a variety of biologically relevant targets have manyshortcomings. The tedious nature, high cost, multi-step character, andlow sensitivity of many of the above-mentioned screening technologiesare shortcomings of the currently available tools. Further, availabletechniques do not always afford the most relevant structuralinformation—the structure of a target in solution, for example. Insteadthey provide insights into target structures that may only exist in thesolid phase. Also, the need for customized reagents and experiments forspecific tasks is a challenge for the practice of current drug discoveryand screening technologies. Current methods also fail to provide aconvenient solution to the need for deconvolution and identification ofactive members of libraries without having to perform tediousre-syntheses and re-analyses of discrete members of pools or mixtures.

[0013] Therefore, methods for the screening and identification ofcomplex chemical libraries especially combinatorial libraries aregreatly needed such that one or more of the structures of both thetarget and ligand, the site of interaction between the target andligand, and the strength of the target-ligand interaction can bedetermined. Further, in order to accelerate drug discovery, new methodsof screening combinatorial libraries are needed to provide ways for thedirect identification of the bioactive members from a mixture and toallow for the screening of multiple biomolecular targets in a singleprocedure. Straightforward methods that allow selective and controlledcleavage of biopolymers, while also analyzing the various fragments toprovide structural information, would be of significant value to thoseinvolved in biochemistry and drug discovery and have long been desired.Also, it is preferred that the methods not be restricted to one type ofbiomolecular target, but instead be applicable to a variety of targetssuch as nucleic acids, peptides, proteins and oligosaccharides.

OBJECTS OF THE INVENTION

[0014] A principal object of the present invention is to provide novelmethods for the determination of the structure of biomolecular targetsand ligands that interact with them and to ascertain the nature andsites of such interactions.

[0015] A further object of the invention is to determine the structuralfeatures of biomolecular targets such as peptides, proteins,oligonucleotides, and nucleic acids such as the primary sequence, thesecondary and folded structures of biopolymers, and higher ordertertiary and quaternary structures of biomolecules that result fromintramolecular and intermolecular interactions.

[0016] Yet another object of the invention is to determine the site(s)and nature of interaction between a biomolecular target and a bindingligand or ligands. The binding ligand may be a “small” molecule, abiomolecule such as a peptide, oligonucleotide or oligosaccharide, anatural product, or a member of a combinatorial library.

[0017] A further object of the invention is to determine the relativebinding affinity or dissociation constant of ligands that bind tobiopolymer targets. Preferably, this gives rise to a determination ofrelative binding affinities between a biopolymer such as an RNA/DNAtarget and ligands e.g. members of combinatorially synthesizedlibraries.

[0018] A further object of the invention is to determine the absolutebinding affinity or dissociation constant of ligands that bind tobiopolymer targets.

[0019] A still further object of the present invention is to provide ageneral method for the screening of combinatorial libraries comprisingindividual compounds or mixtures of compounds against a biomoleculartarget such as a nucleic acid, so as to determine which components ofthe library bind to the target.

[0020] An additional object of the present invention is to providemethods for the determination of the molecular weight and structure ofthose members of a combinatorial library that bind to a biomoleculartarget.

[0021] Yet another object of the invention is to provide methods forscreening multiple targets such as nucleic acids, proteins, and otherbiomolecules and oligomers simultaneously against a combinatoriallibrary of compounds.

[0022] A still further object of the invention is to ascertain thespecificity and affinity of compounds, especially “small” organicmolecules to bind to or interact with molecular interaction sites ofbiological molecules, especially nucleic acids such as RNA. Suchmolecules may be and preferably do form ranked hierarchies of ligandsand potential ligands for the molecular interaction sites, ranked inaccordance with predicted or calculated likelihood of interaction withsuch sites.

[0023] Another object of the present invention is to alleviate theproblem of peak overlap in mass spectra generated from the analysis ofmixtures of screening targets and combinatorial or other mixtures ofcompounds. In a preferred embodiment, the invention provides methods tosolve the problems of mass redundancy in combinatorial or other mixturesof compounds, and also provides methods to solve the problem of massredundancy in the mixture of targets being screened.

[0024] A further object of the invention is to provide methods fordetermining the binding specificity of a ligand for a target incomparison to a control. The present invention facilitates thedetermination of selectivity, the identification of non-specific effectsand the elimination of non-specific ligands from further considerationfor drug discovery efforts.

[0025] The present invention provides, inter alia, a series of newmethods and applications for the determination of the structure andnature of binding of ligands to a wide variety of biomolecular targets.This new approach provides structural information for screeningcombinatorial libraries for drug lead discovery.

SUMMARY OF THE INVENTION

[0026] One aspect of the invention is a method to determine thestructure of biomolecular targets such as nucleic acids using massspectrometry. The method provides not only the primary, sequencestructure of nucleic acid targets, but also information about thesecondary and tertiary structure of nucleic acids, RNA and DNA,including mismatched base pairs, loops, bulges, kinks, and stemstructures. This can be accomplished in accordance with one embodimentby incorporating deoxynucleotide residues or other modified residuesinto an oligoribonucleotide at specific sites followed by selectivecleavage of these hybrid RNA/DNA nucleic acids in a mass spectrometer.It has now been found that electrospray ionization of the nucleic acid,cleavage of the nucleic acid, and subsequent tandem MS^(n) spectrometryaffords a pattern of fragments that is indicative of the nucleic acidsequence and structure. Cleavage is dependent on the sites ofincorporation of the deoxynucleotide or other foreign residues and thesecondary structure of the nucleic acid. This method therefore providesmass spectral data that identifies the sites and types of secondarystructure present in the sequence of nucleic acids.

[0027] When the present methods are performed on a mixture of thebiomolecular target and a ligand or molecule that binds to the target,it is possible to ascertain both the extent of interaction and thelocation of this interaction between ligand and biomolecule. The bindingof the ligand to the biomolecule protects the binding site on thebiomolecule from facile cleavage during mass spectrometry. Therefore,comparison of ESI-MS^(n) mass spectra generated, using this method, forRNA/DNA in the presence and the absence of a binding ligand or drugreveals the location of binding. This altered cleavage pattern isclearly discerned in the mass spectrum and correlated to the sequenceand structure of the nucleic acid. Thus, the absolute binding affinityof the test ligand can be determined by the methods of the presentinvention. Comparison of the abundance of the nucleic acid-ligandnoncovalent complex ion to the abundance of a similar complex iongenerated from a standard compound (such as paromomycin for the 16S RNAA site ) whose binding affinity is known, allows for the determinationof relative binding affinity of the test ligand.

[0028] The methods of this invention can be used for the rapid screeningof large collections of compounds. It is also possible to screenmixtures of large numbers-of compounds that are generated viacombinatorial or other means. When a large mixture of compounds isexposed to a biomolecular target, such as a nucleic acid, a smallfraction of ligands may exhibit some binding affinity to the nucleicacid. The actual number of ligands that may be detected as binders isbased on the concentration of the nucleic acid target, the relativeconcentrations of the components of the combinatorial mixture, and theabsolute and relative binding affinities of these components. The methodis capable of separating different noncovalent complexes, usingtechniques such as selective ion trapping, or accumulation and analyzingeach complex for the structure and identity of the bound ligand usingcollisionally activated dissociation or MS^(n) experiments. The methodsof this invention, therefore, can not only serve as methods to screencombinatorial libraries for molecules that bind to biomolecular targets,but can also provide, in a straightforward manner, the structuralidentity of the bound ligands. In this manner, any mass redundancy inthe combinatorial library does not pose a problem, as the methods canprovide high resolution molecular masses and also able to discerndifferences between the different structures of ligands of identicalmolecular mass using tandem methods.

[0029] In accordance with preferred embodiments, a target biomoleculesuch as an RNA having a molecular interaction site, is presented withone or more ligands or suspected ligands for the interaction site underconditions such that interaction or binding of the ligand to themolecular interaction site can occur. The resulting complex, which maybe of one or even hundreds of individual complexes of ligands with theRNA or other biomolecule, is then subjected to mass spectrometricevaluation in accordance with the invention. “Preparative” massspectrometry can isolate individual complexes which can then befragmented under controlled conditions within the mass spectrometricenvironment for subsequent analysis. In this way, the nature and degree,or absolute binding affinity, of binding of the ligands to the molecularinteraction site can be ascertained. Identification of specific, strongbinding ligands can be made and those selected for use either astherapeutics, agricultural, industrial or other chemicals, or the sameused as lead compounds for subsequent modification into improved formsfor such uses.

[0030] A further application of the present invention is the use of massspectrometric methods for the simultaneous screening of multiplebiomolecular targets against combinatorial libraries or mixtures ofcompounds. This rather complex screening procedure is made possible bythe combined power of the mass spectrometric methods used and the way inwhich the screening is performed. When screening multiple target nucleicacids, for example, mass redundancy is a concern, especially if two ormore targets are of similar sequence composition or mass. This problemis alleviated by the present invention, by using special mass modifying,molecular weight tags on the different nucleic acid targets beingstudied. These mass modifying tags are typically large molecular weight,non-ionic polymers including but not limited to, polyethylene glycols,polyacrylamides and dextrans, that are available in many different sizesand weights, and which may be attached at one or more of many differentpossible sites on nucleic acids. Thus similar nucleic acid targets maybe differentially tagged and now be readily differentiated, in the massspectrum, from one another by their distinctly different mass to chargeratios (m/z signals). Using the methods of this invention, screeningefforts can be significantly accelerated because multiple targets cannow be screened simultaneously against mixtures of large numbers ofcompounds.

[0031] Another related advantage of the methods of this invention is theability to determine the specificity of binding interactions between anew ligand and a biomolecular target. By simultaneously screening atarget nucleic acid, for example, and one or more control nucleic acidsagainst a combinatorial library or a specific ligand, it is possible toascertain, using the methods of this invention, whether the ligand bindsspecifically to only the target nucleic acids, or whether the bindingobserved with the target is reproduced with control nucleic acids and istherefore non-specific.

[0032] The methods of the invention are applicable to the study of awide variety of biomolecular targets that include, but are not limitedto, peptides, proteins, receptors, antibodies, oligonucleotides, RNA,DNA, RNA/DNA hybrids, nucleic acids, oligosaccharides, carbohydrates,and glycopeptides. The molecules that may be screened by using themethods of this invention include, but are not limited to, organic orinorganic, small to large molecular weight individual compounds,mixtures and combinatorial libraries of ligands, inhibitors, agonists,antagonists, substrates, and biopolymers, such as peptides, nucleicacids or oligonucleotides. The mass spectrometric techniques which canbe used in the methods of the invention include, but are not limited to,MS^(n), collisionally activated dissociation (CAD) and collisionallyinduced dissociation (CID) and infrared multiphoton dissociation(IRMPD). A variety of ionization techniques may be used including, butnot limited to, electrospray, MALDI and FAB. The mass detectors used inthe methods of this invention include, but are not limited to, FTICR,ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, andtriple quadrupole. The methods of this invention may also use“hyphenated” techniques such as, but not limited to, LC/MS and CE/MS,all as described more fully hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows the sequence and structure of the 27-mer RNA targetcorresponding to the 16S rRNA A-site.

[0034]FIG. 2 shows the ESI-CID-MS of a 27-mer RNA/DNA hybrid in thepresence and absence of paromomycin.

[0035]FIG. 3 shows the ESI-MS of a 27-mer RNA/DNA hybrid target in thepresence of paromomycin alone (panel a), and in the presence of bothparomomycin and a combinatorial library (panel b).

[0036]FIG. 4 shows the ESI-CID-MS spectrum of a combinatorial librarymember-27mer RNA/DNA hybrid noncovalent complex ion of m/z 1919.0.

[0037]FIG. 5 shows the ESI-MS of a combinatorial library screenedagainst a 27mer RNA/DNA hybrid.

[0038]FIG. 6 shows the ESI-MS-MS analysis of the signal of m/z 1917.8 uarising from the binding of a member of mass 665 from anothercombinatorial library.

[0039]FIG. 7 shows the ESI-MS-MS analysis of the signal of m/z 1934.3 uarising from the binding of a member of mass 720 from a library.

[0040]FIGS. 8 and 9 show graphical representations of the abundances ofw and a-Base ions resulting from (CID) of ions from a DNA:DNA duplex.

[0041]FIGS. 10, 11 and 12 depict MASS analyses to determine the bindingof ligands to a molecular interaction site.

[0042]FIG. 13 depicts high precision ESI-FTICR mass measurement of theinteraction of the 16S A site of an RNA complexed with paromomycin.

[0043]FIG. 14 depicts FTMS spectrum obtained from a mixture of a 16S RNAmodel (10 μM) and a 60-member combinatorial library.

[0044]FIG. 15 depicts an expanded view of the 1863 complex from FIG. 15.

[0045]FIG. 16 depicts mass of a binding ligand determined from astarting library-of compounds.

[0046]FIG. 17 depicts high resolution ESI-FTICR spectrum of the libraryused in FIGS. 15 and 16.

[0047]FIG. 18 depicts use of exact mass measurements and elementalconstraints to determine the elemental composition of an exemplary“unknown” binding ligand.

[0048]FIG. 19 depicts ESI-MS measurements of a solution containing afixed concentration of RNA at different concentrations of ligand.

[0049]FIG. 20 depicts a preferred schematic representation for thedetermination of ligand binding site by tandem mass spectrometry.

[0050]FIG. 21 depicts MASS screening of a 27 member library against a27-mer RNA construct representing the prokaryotic 16S A-site.

[0051]FIG. 22 depicts MS/MS of a 27-mer RNA construct representing theprokaryotic 16S A-site containing deoxyadenosine residues at theparomomycin binding site.

[0052]FIG. 23 depicts MS-MS spectra obtained from a mixture of a 27-merRNA construct representing the prokaryotic 16S A-site containingdeoxyadenosine residues at the paromomycin binding and the 216 membercombinatorial library.

[0053]FIG. 24 depicts secondary structures of the 27 base RNA modelsused in this work corresponding to the 18S (eukaryotic) and 16S(prokaryotic) A-sites.

[0054]FIG. 25 depicts ESI-FTICR spectrum of a mixture of 27-baserepresentations of the 16S A-site with (7 μM) and without (1 μM) an 18atom neutral mass tag attached to the 5-terminus in the presence of 500nM paromomycin.

[0055]FIG. 26 depicts mass spectra from simultaneous screening of 16SA-site and 18S A-site Model RNAs against a mixture of aminoglycosides.

[0056]FIG. 27 depicts sequences and structures for oligonucleotides Rand C.

[0057]FIG. 28A depicts mass spectrum obtained from a mixture of 5 μM Cand 125 nM paromomycin.

[0058]FIG. 28B depicts MS-MS spectrum obtained following isolation of[M-5H]⁵⁻ ions (m/z 1783.6) from uncomplexed C.

[0059]FIG. 28C depicts MS-MS spectrum obtained following isolation of[M-5H]⁵⁻ ions (m/z 1907.5) from C complexed with paromomycin.

[0060]FIG. 29A depicts MS-MS spectrum obtained from a mixture of 10 μM Cand a 216 member combinatorial library following isolation of [M-5H]⁵⁻ions (m/z 1919.0) from C complexed with ligands of mass 676.0±0.6.

[0061]FIG. 29B depicts MS-MS spectrum obtained from a mixture of 10 μM Cand a 216 member combinatorial library following isolation of [M-5H]⁵⁻ions (m/z 1934.3) from C complexed with ligands of mass 753.5±0.6.

[0062]FIG. 30 depicts electrospray ionization Fourier transform ioncyclotron resonance mass spectrometry of a target/putative ligandmixture.

[0063]FIG. 31 shows isotope clusters from the spectrum of FIG. 30.

[0064]FIG. 32 depicts data tabulated and stored in a relationaldatabase.

[0065]FIG. 33 shows an exemplary flow chart for a computer program foreffecting certain methods in accordance with the invention.

SUMMARY OF EXEMPLARY MASS SPECTROMETRIC TECHNIQUES

[0066] Mass spectrometry (MS) is a powerful analytical tool for thestudy of molecular structure and interaction between small and largemolecules. The current state-of-the-art in MS is such that less thanfemtomole quantities of material can be readily analyzed using massspectrometry to afford information about the molecular contents of thesample. An accurate assessment of the molecular weight of the materialmay be quickly obtained, irrespective of whether the sample's molecularweight is several hundred, or in excess of a hundred thousand, atomicmass units or Daltons (Da). It has now been found that mass spectrometrycan elucidate significant aspects of important biological molecules. Onereason for the utility of MS as an analytical tool in accordance withthe invention is the availability of a variety of different MS methods,instruments, and techniques which can provide different pieces ofinformation about the samples.

[0067] One such MS technique is electrospray ionization massspectrometry (ESI-MS) (Smith et al., Anal. Chem., 1990, 62, 882-899;Snyder, in Biochemical and biotechnological applications of electrosprayionization mass, American Chemical Society, Washington, D.C., 1996;Cole, in Electrospray ionization mass spectrometry: fundamentals,instrumentation, Wiley, New York, 1997). ESI produces highly chargeddroplets of the sample being studied by gently nebulizing the samplesolution in the presence of a very strong electrostatic field. Thisresults in the generation of highly charged droplets that shrink due toevaporation of the neutral solvent and ultimately lead to a “Coulombicexplosion” that affords multiply charged ions of the sample material,typically via proton addition or abstraction, under mild conditions.ESI-MS is particularly useful for very high molecular weight biopolymerssuch as proteins and nucleic acids greater than 10 kDa in mass, for itaffords a distribution of multiply-charged molecules of the samplebiopolymer without causing any significant amount of fragmentation. Thefact that several peaks are observed from one sample, due to theformation of ions with different charges, contributes to the accuracy ofESI-MS when determining the molecular weight of the biopolymer becauseeach observed peak provides an independent means for calculation of themolecular weight of the sample. Averaging the multiple readings ofmolecular weight so obtained from a single ESI-mass spectrum affords anestimate of molecular weight that is much more precise than would beobtained if a single molecular ion peak were to be provided by the massspectrometer. Further adding to the flexibility of ESI-MS is thecapability to obtain measurements in either the positive or negativeionization modes.

[0068] In recent years electrospray ionization mass spectrometry(ESI-MS) has grown extensively as an analytical technique due to itsbroad applicability for analysis of macromolecules, including proteins,nucleic acids, and carbohydrates. Bowers, et al., Journal of PhysicalChemistry, 1996,100, 12897-12910; Burlingame, et al., J. Anal. Chem.,1998, 70, 647R-716R; Biemann, Ann. Rev. Biochem., 1992,61,977-1010; andCrain, et al., Curr. Opin. Biotechnol., 1998, 9, 25-34. One of the mostsignificant developments in the field has been the observation, underappropriate solution conditions and analyte concentrations, of specificnon-covalently associated macromolecular complexes that have beenpromoted into the gas-phase intact. Loo, Mass Spectrometry Reviews,1997, 16, 1-23; Smith, et al., Chemical Society Reviews, 1997, 26,191-202; Ens, et al., Standing, K. G. and Chemushevich, I. V. Editors,New Methods for the Study of Biomolecular Complexes (Proceedings of theNATO Advanced Research Workshop, held Jun. 16-20, 1996, in Alberta,Canada In: NATOASISer., Ser. C, 1998; 510; Kluwer, Dordrecht, Neth.,1998. Recent examples include multimeric proteins (Fitzgerald, et al.,Proc. Nat'l. Acad. Sci. USA, 1996, 93,6851-6856), enzyme-ligandcomplexes (Ganguly, et al., Tetrahedron, 1993, 49, 7985-7996),protein-DNA complexes (Cheng, et al., Proc. Nat'l. Acad. Sci. U.S.A.,1996, 93, 7022-7027), multimeric DNA complexes (Griffey, et al., Proc.SPIE-Int. Soc. Opt. Eng., 1997,2985,82-86), and DNA-drug complexes(Gale, et al., JACS, 1994, 116, 6027-6028), the disclosures of which areincorporated herein by reference in their entirety.

[0069] Smith and co-workers have demonstrated that under competitivebinding conditions in solution, ESI-MS measurements of enzyme-ligandmixtures yield gas-phase ion abundances that correlate with measuredsolution-phase dissociation constants (K_(D)). Cheng, et al., JACS,1995, 117, 8859-8860, the disclosure of which is incorporated herein byreference in its entirety. They were able to rank the binding affinitiesof a 256-member library of modified benzenesulfonamide inhibitors tocarbonic anhydrase. Levels of free and bound ligands and substrates canbe quantified directly from their relative abundances as measured byESI-MS and that these measurements can be used to quantitativelydetermine molecular dissociation constants that agree with solutionmeasurements. Jorgensen and co-workers have demonstrated that therelative ion abundance of non-covalent complexes formed between D- andL-tripeptides and vancomycin group antibiotics can be used to measuresolution binding constants. Jorgensen, et al., Anal. Chem., 1998, 70,4427-4432, the disclosure of which is incorporated herein by referencein its entirety. Griffey and co-workers have shown that tandem ESI-MSmethods can be used to determine the binding sites for small moleculesthat bind to RNA targets. Gale, et al., Journal of the American Societyfor Mass Spectrometry, 1995, 6,1154-1164, the disclosure of which isincorporated herein by reference in its entirety.

[0070] Fourier transform ion cyclotron resonance mass spectrometry(FT-ICR MS) can resolve very small mass differences providingdetermination of molecular mass with unparalleled precision andaccuracy. Marshall, et al., Mass Spectrom. Rev., 1998, 17, 1-35. Becauseeach small molecule with a unique elemental composition carries anintrinsic mass label corresponding to its exact molecular mass,identifying closely related library members bound to a macromoleculartarget requires only a measurement of exact molecular mass. The targetand potential ligands do not require radiolabeling, fluorescent tagging,or deconvolution via single compound re-synthesis. Furthermore,adjustment of the concentration of ligand and target allows ESI-MSassays to be run in a parallel format under competitive ornon-competitive binding conditions. Signals can be detected fromcomplexes with dissociation constants ranging from <10 riM to ˜100 mM.

[0071] Small molecules that bind to structured regions of RNA canexhibit therapeutic effects. For example, aminoglycoside antibioticsinhibit bacterial growth by disrupting essential RNA-protein and RNA-RNAinteractions. De Stasio, et al, EEMBO J, 1989, 8, 1213-6 and Bryan, L.E. In New dimensions in antimicrobial therapy; Root, R. K., Sande, M.A., Eds., Churchill Livingstone, New York, 1984; Vol. 1, pp 17-35.Paromomycin, one of the most widely studied aminoglycosides, binds tothe decoding region of the prokaryotic 16S rRNA (the A-site) with a ˜200nM KD and induces misreading of the genetic code during translation.Wong, et al., Chem. Biol., 1998, 5, 397-406. However, the features ofthe interaction between RNAs and aminoglycosides that provide bindingspecificity are poorly characterized. In this work we employ ESI-FTICRto detect specific interactions between two closely related model RNAconstructs corresponding to the decoding sites of the prokaryotic andeukaryotic ribosomes and individual members of a collection ofaminoglycoside antibiotics.

[0072] Matrix-Assisted Laser DesorptionlIonization Mass Spectrometry(MALDI-MS) is another method that can be used for studying biomolecules(Hillenkamp et al., Anal. Chem., 1991, 63, 1193A-1203A). This techniqueionizes high molecular weight biopolymers with minimal concomitantfragmentation of the sample material. This is typically accomplished viathe incorporation of the sample to be analyzed into a matrix thatabsorbs radiation from an incident UV or IR laser. This energy is thentransferred from the matrix to the sample resulting in desorption of thesample into the gas phase with subsequent ionization and minimalfragmentation. One of the advantages of MALDI-MS over ESI-MS is thesimplicity of the spectra obtained as MALDI spectra are generallydominated by singly charged species. Typically, the detection of thegaseous ions generated by MALDI techniques, are detected and analyzed bydetermining the time-of-flight (TO) of these ions. While MALDI-TOF MS isnot a high resolution technique, resolution can be improved by makingmodifications to such systems, by the use of tandem MS techniques, or bythe use of other types of analyzers, such as Fourier transform (FT) andquadrupole ion traps.

[0073] Fourier transform mass spectrometry (FTMS) is an especiallyuseful analytical technique because of its ability to make massmeasurements with a combination of accuracy and resolution that issuperior to other MS detection techniques, in connection with ESI orMALDI ionization (Amster, J. Mass Spectrom., 1996,31, 1325-1337).Furtheritmaybe used to obtain high resolution mass spectra of ionsgenerated by any of the other ionization techniques. The basis for FTMSis ion cyclotron motion, which is the result of the interaction of anion with a unidirectional magnetic field. The mass-to-charge ratio of anion (m/q or m/z) is determined by a FTMS instrument by measuring thecyclotron frequency of the ion. The insensitivity of the cyclotronfrequency to the kinetic energy of an ion is one of the fundamentalreasons for the very high resolution achievable with FTMS. FTMS is anexcellent detector in conventional or tandem mass spectrometry, for theanalysis of -ions generated by a variety of different ionization methodsincluding ESI and MALDI, or product ions resulting from collisionallyactivated dissociation (CAD).

[0074] Collisionally activated dissociation (CAD), also known ascollision induced dissociation (CID), is a method by which analyte ionsare dissociated by energetic collisions with neutral or charged species,resulting in fragment ions which can be subsequently mass analyzed. Massanalysis of fragment ions from a selected parent ion can provide certainsequence or other structural information relating to the parent ion.Such methods are generally referred to as tandem mass spectrometry (MSor MS/MS) methods and are the basis of the some of MS based biomolecularsequencing schemes being employed today.

[0075] FTICR-MS, like ion trap and quadrupole mass analyzers, allowsselection of an ion that may actually be a weak non-covalent complex ofa large biomolecule with another molecule (Marshall and Grosshans, Anal.Chem., 1991, 63, A215-A229; Beu et al., J. Am. Soc. Mass Spectrom.,1993, 4, 566-577; Winger et al., J. Am. Soc. Mass Spectrom., 1993, 4,566-577); (Huang and Henion, Anal. Chem., 1991, 63, 732-739), orhyphenated techniques such as LC-MS (Bruins, Covey and Henion, Anal.Chem., 1987, 59, 2642-2646 Huang and Henion, J. Am. Soc. Mass Spectrom.,1990, 1, 158-65; Huang and Henion, Anal. Chem., 1991, 63, 732-739) andCE-MS (Cai and Henion, J. Chromatogr., 1995, 703, 667-692) experiments.FTICR-MS has also been applied to the study of ion-molecule reactionpathways and kinetics.

[0076] So-called “Hyphenated” techniques can be used for structureelucidation because they provide the dual features of separation andmass detection. Such techniques have been used for the separation andidentification of certain components of mixtures of compounds such asthose isolated from natural products, synthetic reactions, orcombinatorial chemistry. Hyphenated techniques typically use aseparation method as the first step; liquid chromatography methods suchas HPLC, microbore LC, microcapillary LC, or capillary electrophoresisare typical separation methods used to separate the components of suchmixtures. Many of these separation methods are rapid and offer highresolution of components while also operating at low flow rates that arecompatible with MS detection. In those cases where flow rates arehigher, the use of ‘megaflow’ ESI sources and sample splittingtechniques have facilitated their implementation with on-line massspectrometry. The second stage of these hyphenated analytical techniquesinvolves the injection of separated components directly into a massspectrometer, so that the spectrometer serves as a detector thatprovides information about the mass and composition of the materialsseparated in the first stage. While these techniques are valuable fromthe standpoint of gaining an understanding of the masses of the variouscomponents of multicomponent samples, they are incapable of providingstructural detail. Some structural detail, however, may be ascertainedthrough the use of tandem mass spectrometry, e.g., hydrogen/deuteriumexchange or collision induced disassociation.

[0077] Typically, tandem mass spectrometry (MSn) involves the coupleduse of two or more stages of mass analysis where both the separation anddetection steps are based on mass spectrometry. The first stage is usedto select an ion or component of a sample from which further structuralinformation is to be obtained. This selected ion is then fragmented by(CID) or photodissociation. The second stage of mass analysis is thenused to detect and measure the mass of the resulting fragments orproduct ions. The advent of FTICR-MS has made a significant impact onthe utility of tandem, MS^(n) procedures because of the ability of FTICRto select and trap specific ions of interest and its high resolution andsensitivity when detecting fragment ions. Such ion selection followed byfragmentation routines can be performed multiple times so as toessentially completely dissect the molecular structure of a sample. Atwo-stage tandem MS experiment would be called a MS-MS experiment whilean n-stage tandem MS experiment would be referred to as a MS^(n)experiment. Depending on the complexity of the sample and the level ofstructural detail desired, MS^(n) experiments at values of n greaterthan 2 may be performed.

[0078] Ion trap-based mass spectrometers are particularly well suitedfor such tandem experiments because the dissociation and measurementsteps are temporarily rather than spatially separated. For example, acommon platform on which tandem mass spectrometry is performed is atriple quadrupole mass spectrometer. The first and third quadrupolesserve as mass filters while the second quadrupole serves as a collisioncell for CAD. In a trap based mass spectrometer, parent ion selectionand dissociation take place in the same part of the vacuum chamber andare effected by control of the radio frequency wavelengths applied tothe trapping elements and the collision gas pressure. Hence, while atriple quadrupole mass analyzer is limited to two stages of massspectrometry (i.e. MS/MS), ion trap-based mass spectrometers can performMS^(n) analysis in which the parent ion is isolated, dissociated, massanalyzed and a fragment ion of interest is isolated, furtherdissociated, and mass analyzed and so on. A number of MS⁴ procedures andhigher have appeared in the literature in recent years and can be usedhere. (Cheng et al., Techniques in Protein Chemistry, VII, pp. 13-21).

[0079] ESI and MALDI techniques have found application for the rapid andstraightforward determination of the molecular weight of certainbiomolecules (Feng and Konishi, Anal. Chem., 1992, 64, 2090-2095;Nelson, Dogruel and Williams, Rapid Commun. Mass Spectrom., 1994, 8,627-631). These techniques have been used to confirm the identity andintegrity of certain biomolecules such as peptides, proteins,oligonucleotides, nucleic acids,, glycoproteins, oligosaccharides andcarbohydrates. Further, these MS techniques have found biochemicalapplications in the detection and identification ofpost-translationalmodifications on proteins. Verification of DNA and RNA sequences thatare less than 100 bases in length has also been accomplished using ESIwith FTMS to measure the molecular weight of the nucleic acids (Littleet al, Proc. Natl. Acad. Sci. USA, 1995, 92, 2318-2322).

[0080] ESI tandem MS has been used for the study of high molecularweight proteins, for peptide and protein sequencing, identification ofpost-translational modifications such as phosphorylation, sulfation orglycosylation, and for the study of enzyme mechanisms (Rossomando etal., Proc. Natl. Acad. Sci. USA, 1992, 89, 5779-578; Knight et al.,Biochemistry, 1993, 32, 2031-2035). Covalent enzyme-intermediate orenzyme-inhibitor complexes have been detected using ESI and analyzed byESI-MS to ascertain the site(s) of modification on the enzyme. Theliterature has shown examples of protein sequencing where the multiplycharged ions of the intact protein are subjected to collisionallyactivated dissociation to afford sequence informative fragment ions(Light-Wahl et al., Biol. Mass Spectrom., 1993, 22, 112-120). ESI tandemMS has also been applied to the study of oligonucleotides and nucleicacids (Ni et al., Anal. Chem., 1996, 68, 1989-1999; Little, Thannhauserand McLafferty, Proc. Natl. Acad. Sci., 1995, 92, 2318-2322).

[0081] While tandem ESI mass spectra of oligonucleotides are oftencomplex, several groups have successfully applied ESI tandem MS to thesequencing of large oligonucleotides (McLuckey, Van Berkel and Glish, J.Am. Soc. Mass Spectrom., 1992,3,60-70; McLuckey and Habibigoudarzi,J.Am. Chem. Soc.,1993,115,12085-12095; Little et al., J.Am. Chem. Soc.,1994, 116, 4893-4897). General rules for the principal dissociationpathways of oligonucleotides, as formulated by McLuckey (McLuckey, VanBerkel and Glish, J. Am. Soc. Mass Spectrom., 1992, 3, 60-70; Mcluckeyand Habibigoudarzi, J. Am. Chem. Soc., 1993, 115, 12085-12095), haveassisted interpretation of mass spectra of oligonucleotides, and includeobservations of fragmentation such as, for example, the stepwise loss ofbase followed by cleavage of the 3′—C—O bond of the relevant sugar.Besides the use of ESI with tandem MS for oligonucleotide sequencing,two other mass spectrometric methods are also available: mass analysisof products of enzymatic cleavage of oligonucleotides (Pieles et al.,Nucleic Acids Res., 1993, 21, 3191-3196; Shaler et al., Rapid Commun.Mass Spectrom., 1995, 9, 942-947; Glover et al., Rapid Commun. MassSpectrom., 1995, 9, 897-901), and the mass analysis of fragment ionsarising from the initial ionization/desorption event, without the use ofmass selection techniques (Little et al., Anal. Chem., 1994,66,2809-2815; Nordhoffet al., J. Mass Spectrom., 1995, 30,99-112; Littleet al., J. Am. Chem. Soc., 1994,116,4893-4897; Little and McLafferty, J.Am. Chem. Soc., 1995, 117, 6783-6784). While determining the sequence ofdeoxyribonucleic acids (DNA) is possible using ESI-MS and CID techniques(McLuckey, Van Berkel and Glish, J. Am. Soc. Mass Spectrom., 1992, 3,60-70; McLuckey and Habibigoudarzi, J. Am. Chem. Soc.,1993,115,12085-12095), the determination of RNA sequence is much moredifficult. Thus while small RNA, such as 6-mers, have been sequenced(McCloskey et al., J. Am. Chem. Soc., 1993,115,12085-1095), larger RNAhave been difficult to sequence using mass spectrometry.

[0082] Electrospray mass spectrometry has been used to study biochemicalinteractions of biopolymers such as enzymes, proteins and nucleic acidswith their ligands, receptors, substrates or inhibitors. Whileinteractions that lead to covalent modification of the biopolymer havebeen studied for some time, those interactions that are of anon-covalent nature have been particularly difficult to study heretoforeby methods other than kinetic techniques. It is now possible to yieldinformation on the stoichiometry and nature of such non-covalentinteractions from mass spectrometry. MS can provide information aboutthe interactions between biopolymers and other molecules in the gasphase; however, experiments have demonstrated that the data so generatedcan be reflective of the solution phase phenomena from which the massspectra were generated.

[0083] ESI is a gentle ionization method that results in no significantmolecular fragmentation and preserves even weakly bound complexesbetween biopolymers and other molecules so that they are detected intactwith mass spectrometry. A variety of non-covalent complexes ofbiomolecules have been studied using ESI-MS and reported in theliterature (Loo, Bioconjugate Chemistry, 1995, 6,644-665; Smith et al.,J. Biol. Mass Spectrom. 1993, 22,493-501; Li et al., J. Am. Chem. Soc.,1993, 115,8409-8413). These include the peptide-protein complexes(Busman et al., Rapid Commun. Mass Spectrom., 1994, 8,211-216; Loo,Holsworth and Root-Bernstein, Biol. Mass Spectrom., 1994,23,6-12;Anderegg and Wagner, J. Am. Chem. Soc., 1995, 117, 1374-1377;Baczynskyj, Bronson and Kubiak., Rapid Commun. Mass Spectrom., 1994,8,280-286), interactions of polypeptides and metals (Loo, Hu and Smith,J. Am. Soc. Mass Spectrom., 1994, 5, 959-965; Hu and Loo, J. MassSpectrom.,1995,30,1076-1079;Witkowska et al., J. Am. Chem. Soc.,1995,117,3319-3324; Lane et al., J. Cell Biol., 1994, 125,929-943),protein-small molecule complexes (Ganem and Henion, ChemTracts-Org.Chem., 1993, 6, 1-22; Henion et al., Ther. Drug Monit., 1993, 15,563-569; Baca and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993), thestudy of the quaternary structure of multimeric proteins (Baca and Kent,J. Am. Chem. Soc., 1992, 114., 3992-3993; Light-Wahl, Schwartz andSmith, J. Am. Chem. Soc., 1994,116,5271-5278; Loo, J. Mass Spectrom.,1995, 30,180-183), and the study of nucleic acid complexes (Light-Wahlet al., J. Am. Chem. Soc., 1993, 115, 803-804; Gale et al., J. Am. Chem.Soc., 1994, 116, 6027-6028; Goodlett et al., Biol. Mass Spectrom.,1993,22,181-183; Ganem, Li and Henion, Tet. Lett., 1993, 34, 1445-1448;Doctycz et al., Anal. Chem., 1994, 66, 3416-3422; Bayer et al., Anal.Chem., 1994, 66, 3858-3863; Greig et al., J. Am. Chem. Soc., 1995, 117,10765-766).

[0084] While data generated and conclusions reached from ESI-MS studiesfor weak non-covalent interactions generally reflect, to some extent,the nature of the interaction found in the solution-phase, it has beenpointed out in the literature that control experiments are necessary torule out the possibility of ubiquitous non-specific interactions (Smithand Light-Wahl, Biol. Mass Spectrom., 1993,22,493-501). Some haveapplied the use of ESI-MS and MALDI-MS to the study of multimericproteins for the gentleness of the electrospray/desorption processallows weakly bound complexes, held together by hydrogen bonding,hydrophobic and/or ionic interactions, to remain intact upon transfer tothe gas phase. The literature shows that not only do ESI-MS data fromgas-phase studies reflect the non-covalent interactions found insolution, but that the strength of such interactions may also bedetermined. The binding constants for the interaction of various peptideinhibitors to src SH2 domain protein, as determined by ESI-MS, werefound to be consistent with their measured solution phase bindingconstants (Loo, Hu and Thanabal, Proc. 43^(rd) ASMS Conf. on MassSpectrom. and Allied Topics, 1995). ESI-MS has also been used togenerate Scatchard plots for measuring the binding constants ofvancomycin antibiotics with tripeptide ligands (Lim et al., J. MassSpectrom., 1995, 30, 708-714).

[0085] Similar experiments have been performed to study non-covalentinteractions of nucleic acids. Both ESI-MS and MALDI-MS have beenapplied to study the non-covalent interactions of nucleic acids andproteins. While MALDI does not typically allow for survival of an intactnon-covalent complex, the use of crosslinking methods to generatecovalent bonds between the components of the complex allows for its usein such studies. Stoichiometry of interaction and the sites ofinteraction have been ascertained for nucleic acid-protein interactions(Jensen et al., Rapid Commun. Mass Spectrom., 1993, 7, 496-501; Jensenet al., 42^(nd) ASMS Conf. on Mass Spectrom. and Allied Topics, 1994,923). The sites of interaction are typically determined by proteolysisof either the non-covalent or covalently crosslinked complex (Jensen etal., Rapid Commun. Mass Spectrom., 1993, 7,496-501; Jensen et al.,42^(nd) ASMS Conf. on Mass Spectrom. and Allied Topics, 1994, 923; Cohenet al., Protein Sci., 1995, 4, 1088-1099). Comparison of the massspectra with those generated from proteolysis of the protein aloneprovides information about cleavage site accessibility or protection inthe nucleic acid-protein complex and, therefore, information about theportions of these biopolymers that interact in the complex.

[0086] Electrospray mass spectrometry has also been effectively used forthe determination of binding constants of noncovalent macromolecularcomplexes such as those between proteins and ligands, enzymes andinhibitors, and proteins and nucleic acids. Greig et al. (J. Am. Chem.Soc., 1995, 117, 10765-10766) have reported the use of ESI-MS todetermine the dissociation constants (K_(D)) for oligonucleotide-bovineserum albumin (BSA) complexes. The K_(D) values determined by ESI-MSwere reported to match solution KD values obtained using capillaryelectrophoresis.

[0087] Cheng et al. (J. Am. Chem. Soc., 1995, 117, 8859-8860) havereported the use of ESI-FTICR mass spectrometry as a method to determinethe structure and relative binding constants for a mixture ofcompetitive inhibitors of the enzyme carbonic anhydrase. Using a singleESI-FTICR-MS experiment these researchers were able to ascertain therelative binding constants for the noncovalent interactions betweeninhibitors and the enzyme by measuring the relative abundances of theions of these noncovalent complexes. Further, the K_(D)s so determinedfor these compounds paralleled their known binding constants insolution. The method was also capable of identifying the structures oftight binding ligands from small mixtures of inhibitors based on thehigh resolution capabilities and multistep dissociation massspectrometry afforded by the FTICR technique. In a related study, Gao etal. (J. Med. Chem., 1996, 39, 1949-55) have reported the use ofESI-FTICR-MS to screen libraries of soluble peptides in a search fortight binding inhibitors of carbonic anhydrase II. Simultaneousidentification of the structure of a tight binding peptide inhibitor anddetermination of its binding constant was performed. The bindingaffinities determined from mass spectral ion abundance were found tocorrelate well with those determined in solution experiments. Further,the applicability of this technique to drug discovery efforts is limitedby the lack of information generated with regards to sites and mode ofsuch noncovalent interactions between a protein and ligands.

[0088] Also, these methods discuss, and appear to be limited to, thestudy of ligand interactions with proteins. The suitability of thismethod of mass spectrometric analysis of binding and dissociationconstants for the study of noncovalent interactions of oligonucleotides,nucleic acids, such as RNA and DNA, and other biopolymers has not beendescribed in the literature.

[0089] The drug discovery process has recently been revolutionized bythe introduction of high throughput synthesis and combinatorialchemistry which afford collections and mixtures of large numbers ofsynthetic compounds for the purpose of screening for biologicalactivity. Such large mixtures and pools of compounds pose significantchallenges for the bioassay and analytical scientist. The analyticalchallenge is two-fold: separation of the active component of a mixture,and the identification of its structure. A variety of separation methodsare available, including LC, HPLC, and CE. However, from the standpointof separating biologically active components from a mixture of one ormore targets with a combinatorial library necessitates the use anddevelopment of methods that select for and separate the complex (usuallynoncovalent) between the ligands and the target. Affinity column methodshave been used to selectively isolate and subsequently analyze bindingcomponents of mixtures of compounds. For example, Kassel et al. (Kasselet al., Techniques in Protein Chemistry VI, J. Crabb, Ed., AcademicPress, San Diego, 1995, 39-46) have used an immobilized src SH2 domainprotein column to separate and then analyze by HPLC-ESI-MS the structureof high affinity binding phosphopeptides.

[0090] A similar technique, ACE-ESI-MS, uses affinity capillaryelectrophoresis to accomplish the separation of noncovalent complexesformed upon mixing a biomolecular target with a combinatorial library ormixture of compounds. The receptor is typically incorporated into thecapillary so that those ligands present in the combinatorial mixtureinteract with the target and are retained or slowed down within thecapillary. Once separated, these noncovalent complexes are analyzedon-line by ESI-MS to ascertain the structures of the complexes and boundcomponents. This method incorporates into one, the two steps that werepreviously performed separately: the compound/noncovalent complexselection, as has previously been demonstrated for vancomycin (Chu etal., Acc. Chem. Res., 1995, 28, 461-468; Chu et al., J. Org. Chem.,1993,58,648-52) and the step of compound identification (Cai and Henion,J. Chromatogr., 1995, 703, 667-692). For example, ACE-ESI-MS has beenapplied to mixtures of vancomycin with peptide libraries (Chu et al., J.Am. Chem. Soc., 1996, 118, 7827-35) to allow rapid screening ofnoncovalent complexes formed, and the identification of peptides thatbind to vancomycin.

[0091] Another method for the separation and identification of activecomponents from combinatorial libraries is the use of size-exclusionchromatography (SEC) followed by LC/MS or CE/MS analysis. Size exclusionis a simple yet powerful method to separate a biopolymer target and itscomplexes with small molecules members of a combinatorial library. Onceisolated by SEC, these complexes are dissociated, under denaturingsolution conditions, and finally the binding ligands are analyzed bymass spectrometry. This method has been applied to the identification ofhigh affinity ligands for human serum albumin (HSA) from combinatoriallibrary of small molecules (Dunayevskiy et al., Rapid Commun. MassSpectrom., 1997,11, 1178-84).

[0092] Bio-affinity characterization mass spectrometry (BACMS) is yetanother method for the characterization of noncovalent interactions ofmixtures of ligands and biomolecular targets (Bruce et al., RapidCommun. Mass Spectrom., 1995, 9, 644-50). BACMS involves theelectrospray ionization of a solution containing both the affinitytarget and a mixture of ligands (or a combinatorial library), followedby trapping of all the ionic species in the FTICR ion-trap. Thecomplexes of interest are then identified in the mass spectrum andisolated by selected-ion accumulation. This is followed by low energydissociation or ‘heating’ to separate the high binding affinity ligandspresent in the complex. Finally, collisionally activated dissociation(CAD) is used to provide structural information about the high bindingaffinity ligand. The greatest advantage of BACMS is that thetime-consuming techniques usually needed for the study of libraries,such as affinity chromatography, using solid supports for separation andpurification of the complexes, followed by analysis to characterize theselected ligands, are all combined into one FTICR-MS experiment. Todate, BACMS has only been applied to the study of protein targets.

[0093] None of the foregoing methods, however, have demonstratedapplicability to a variety of biomolecular targets. Further, suchmethods do not provide rapid determination of the site of interactionbetween a combinatorially derived ligand and biopolymer.

[0094] Tandem mass spectrometry, as performed using electrosprayionization (ESI) on FTICR, triple quadrupole, or ion-trap massspectrometers, has been found to be a powerful tool for determining thestructure of biomolecules. It is known in the art that both small andlarge (>3000 kbase) RNA and DNA may be transferred from solution intothe gas phase as intact ions using electrospray techniques. Further itis known, to those skilled in the art that these ions retain some degreeof their solution structures as ions in the gas phase; this isespecially useful when studying noncovalent complexes of nucleic acidsand proteins, and nucleic acids and small molecules by massspectrometric techniques.

SUMMARY OF CERTAIN PREFERRED EMBODIMENTS

[0095] Studies have demonstrated that oligonucleotides and nucleic acidsobey certain fragmentation patterns during collisionally induceddissociation (CID), and that these fragments and patterns can be used todetermine the sequence of the nucleic McLuckey, Van Berkel and Glish, J.Am. Soc. Mass Spectrom., 1992,3,60-70; Mcluckey and Habibigoudarzi, J.Am. Chem. Soc., 1993, 115, 12085-12095). Electrospray ionizationproduces several multiply charged ions of the parent nucleic acid,without any significant fragmentation of the nucleic acid. Typically, asingle charge state of the nucleic acid is isolated using a triplequadrupole ion trap, or ion cyclotron resonance (ICR) device. This ionis then excited and allowed to collide with a neutral gas such ashelium, argon or nitrogen so as to afford cleavage of certain bonds inthe nucleic acid ion, or excited and fragmented with a laser pulse.Typically, two series of fragment ions are found to be formed: thea-Base series, and the w-series.

[0096] The series of a-Base fragments originates from initial cleavageof the glycosidic bond by simultaneous abstraction of a C-2′ proton,followed by the elimination of the 3′-phosphategroup and the C-4′proton. This fragmentation scheme results in a residual furan attachedto the 3′-phosphate and affords a series of a-Base fragments whosemasses increase sequentially from the 5′-terminus of the nucleic acid.Measurement of the masses of these collisionally induced fragmentstherefore affords the determination of the sequence of the nucleic acidin the 5′ to 3′ direction. The w series of fragments is generated viacleavage of the nucleic acid in a manner that leaves a 5′phosphateresidue on each fragment. Thus monitoring the masses of w-seriesfragments allows determination of the sequence of the nucleic acid inthe 3′ to 5′ direction. Using the sequence information generated fromboth series of fragments the sequence of deoxyribonucleic acids (DNA)may be ascertained. Obtaining similar mass spectrometric information forribonucleic acids (RNA), is a much more difficult task. Collisionallyinduced dissociation (CID) of RNA is much less energetically favoredthan is the case for DNA because of the greater strength of theglycosidic bond in RNA. Hence, while small RNA such as 6-mers have beensequenced using CID MS, the sequencing of larger RNA has not beengenerally successful using tandem MS.

[0097] Determination of the structure of biomolecules, such as proteinsand nucleic acids, may be attempted using solution biochemical cleavagefollowed by mass spectrometry. However, these methods are cumbersome andnot always successful in that several biochemical cleavage andseparation steps need to be performed prior to MS analysis of thecleaved products. Also, the level of information provided with regardsto secondary and tertiary structure of biomolecules is limited. Methodsavailable in the scientific literature are therefore greatly limited interms of the sequence and structural information they provide forbiomolecules and biomolecular targets.

[0098] One aspect of the present invention provides methods fordetermining the structure of biomolecular targets such as nucleic acidsusing mass spectrometry. The structure of nucleic acids, especially RNA,which is often difficult to ascertain, is readily determined using themethods of this invention. The structure of a nucleic acid is determinedfrom the fragmentation pattern observed in MS^(n) experiments. Directedfragmentation of RNA is facilitated by the selective incorporation ofdeoxynucleotides or other nucleosidic residues at specific residuelocations in the nucleic acid sequence. During CID of such RNA/DNAchimeric nucleic acids, cleavage is facilitated at the sites wheredeoxynucleotides or the other non-native residues were incorporated.Cleavage is also influenced by the local secondary and tertiarystructure of the biomolecule. Therefore, the cleavage patterns observedfrom a RNA/DNA hybrid reveals the local structure of the nucleic acid,including mismatched base pairs, bulged regions and other features.

[0099] Since exposed deoxynucleotide residues are known to besusceptible to CID cleavage in MS experiments, the systematicincorporation of such residues into RNA allows the systematicexploration of the local structure of RNA. Using this embodiment of theinvention, it is possible to determine the secondary and tertiarystructure of nucleic acids, including features such as mismatched basepairs, loops, bulges, and kink and stem structures.

[0100] Determination of the structure of an RNA may be accomplished,using exemplary methods of the invention, as follows. An RNA whosestructure is to be determined is synthesized using an automated nucleicacid synthesizer. During RNA synthesis, deoxynucleotides are selectivelyincorporated into the sequence at specific sites where the structure isto be probed. This RNA/DNA chimeric nucleic acid, which is sensitized tocollisional activation, is now used for sequence and structuredetermination using tandem MS experiments. ESI-MS, followed by trappingof selected ions and subsequent CID of each ion, affords information asto which positions of the nucleic acid hybrid are disordered (or notparticipating in a higher order structure) and, therefore, available forcleavage. A systematic pattern of deoxynucleotide incorporation into thesequence of the test RNA allows a systematic, mass spectrometricassessment of structure in a certain area of the nucleic acid, or forthe entire nucleic acid. Other modified nucleic acid residues may beused instead of DNA. This, chemically modified nucleic acid subunitssuch as Z¹-modified, e.g. 2¹-O-Alkyl, base-modified, backbone modifiedor other residues may serve. Such residues will permit assessment of DNAas well as RNA.

[0101] The present invention also provides methods for the determinationof the site and nature of interactions between a biomolecular target anda binding ligand. This is information of critical value to the processof drug discovery. Current methods of biomolecular screening do notprovide a straightforward means of also determining the nature of theinteraction between a binding ligand and the biomolecular target.Information such as the stoichiometry and binding affinity of theinteraction often needs to be ascertained from additional biochemicalassays, thus slowing down and increasing the cost of drug discovery. Itis often the case that binding of a drug or ligand to a biomoleculartarget, such as a nucleic acid, may lead to a change in conformation ofthe biomolecule to a different structure. This, too, may contribute toprotection of the biomolecule from cleavage.

[0102] The present invention provides convenient methods for determiningthe site or sites on a biomolecular target where a binding ligandinteracts. This is accomplished based on the knowledge thatcollisionally activated dissociation (CID or CAD) of a noncovalent,biomolecule-ligand complex may be performed such that cleavage of thecomplex occurs only at exposed sites of the biomolecules. Thus cleavagesites present on the biomolecule that are involved in binding with theligand are protected because of the increased structural order from thebinding event during CID. ESI-MS^(n) Spectra generated using thismethod, in the presence and absence of a binding ligand (or drug), willreveal differential fragmentation patterns due to ligand inducedprotection of cleavage sites. Comparison of the mass spectra generatedin the presence and absence of a binding ligand will, therefore, revealthe positions in the biomolecular sequence where the interactionsbetween ligand and biomolecule are occurring.

[0103] These methods for determining the sites of interaction between abinding ligand and a biomolecular target are broadly applicable. Thebiomolecular targets that may be studied using this method include, butare not limited to, peptides, proteins, antibodies, oligonucleotides,RNA, DNA, other nucleic acids, glycopeptides, and oligosaccharides. Itis preferred that the biomolecular target be a nucleic acid. It isfurther preferred that the biomolecular target be a chimeric RNA/DNAnucleic acid, synthesized to selectively incorporate deoxynucleotides,(or other residues) in the sequence at specific locations. The bindingligand may be one of the groups of molecules including, but not limitedto, organic or inorganic, small to large molecular weight individualcompounds, mixtures and combinatorial libraries of ligands, inhibitors,agonists, antagonists, substrates, and biopolymers, such as peptides oroligonucleotides.

[0104] Determination of the sites on an RNA target where interactionoccurs with a binding ligand may be accomplished as follows. An RNAtarget that is to be studied as a biomolecular target is prepared usingan automated synthesizer, and selectively incorporating deoxynucleotidesinto the sequence at specific sites. An aliquot of this RNA/DNA chimericis used directly for ESI-MS, followed by CID analysis of selectivelyaccumulated ions, to establish the native structure and cleavagepatterns of this biomolecular target. A second aliquot of the RNA/DNAchimeric is mixed with a solution of a drug or ligand that is known tobind to the biomolecular target. The target and ligand are anticipatedto interact in solution to form a noncovalent complex. Subjecting thissolution of the noncovalent biomolecule-ligand complex to the method ofthis invention leads to ionization of the complex with a retention ofthe noncovalent interactions and binding stoichiometries. CID of thecomplex then leads to cleavage of the biomolecule sequence atfragmentation sites that are exposed. Sites where fragmentation wouldotherwise occur, but which are involved in binding the ligand to thebiomolecule, are protected, such that cleavage at or near these sites isprevented during the CID stage. The differences in the fragmentationpatterns of the biomolecule when, subjected to the methods of thisinvention in the presence and absence of binding ligand indicate thesite(s) on the biomolecule that is protected and, therefore, areinvolved in binding the ligand.

[0105] Likewise, a systematic pattern of deoxynucleotide incorporationinto the sequence of the test RNA will allow for a systematic massspectrometric assessment of binding sites and interactions in a certainarea of the nucleic acid, or for the entire nucleic acid, using themethod of this invention. This invention, therefore, also provides a newmethod of ‘footprinting’ biomolecular targets especially nucleic acids.This footprinting by mass spectrometry is a straightforward method formapping the structure of biomolecular targets and the sites ofinteractions of ligands with these targets.

[0106] The nature of interactions between the binding ligand and abiomolecular target are also readily studied using the method of thisinvention. Thus, the stoichiometry and absolute and relativedissociation constant of the biomolecule-ligand noncovalent complex isreadily ascertained using the method of this invention. The ratio of thenumber of ligand molecules and the number of biomolecular receptorsinvolved in the formation of a noncovalent biomolecule-ligand complex isof significant importance to the biochemist and medicinal chemist.Likewise, the strength of a noncovalent complex, or the binding affinityof the ligand for the biomolecular target, is of significance because itprovides an indication of the degree of complementarity between theligand and the biomolecule. Also, the determination of this bindingaffinity is important for the rank ordering of different ligands so asto provide structure-activity relationships for a series of ligands, andto facilitate the design of stronger binding ligands for a particularbiomolecular target.

[0107] The methods of the present invention are also capable ofdetermining both the binding stoichiometry and affinity of a ligand forthe biomolecular target being screened in a screening study.Electrospray ionization is known to retain to a significant degree, thesolution phase structures of biomolecules and their noncovalentcomplexes in the gaseous ions it generates. Thus, determination of thestoichiometry of noncovalent complexes simply needs data on the massesof the ligand, biomolecular target and the noncovalentbiomolecule-ligand complex. The data needed to accomplish thisdetermination is actually available from the mass spectrometryexperiment that may be performed to determine the structure and site ofbinding of a ligand to the biomolecular target. Based on the knowledgeof the structure and sequence of the target biomolecule, MS analysis ofthe biomolecule-ligand complex reveals the number of ligand and targetmolecules present in the noncovalent complex. If the noncovalent complexion observed from the mass spectrum is of an m/z equal to that expectedfrom the addition of the m/z values of one molecule each of the targetbiomolecule and ligand, then the noncovalent complex must be formed froma 1:1 interaction between the biomolecule and ligand. Simplemathematical operations on the molecular weight and charges of thetarget and ligand can likewise determine higher levels of interactionsbetween ligand and biomolecule. The high resolution of a FTICR massspectrometer allows direct identification of the bound ligand based onexact measurement of the molecular mass of the complex relative tounbound nucleic acid.

[0108] The use of mass spectrometry, in accordance with this inventioncan provide information on not only the mass to charge ratio of ionsgenerated from a sample, but also the relative abundance of such ions.Under standardized experimental conditions, it is therefore possible tocompare the abundance of a noncovalent biomolecule-ligand complex ionwith the ion abundance of the noncovalent complex formed between abiomolecule and a standard molecule, such as a known substrate orinhibitor. Through this comparison, binding affinity of the ligand forthe biomolecule, relative to the known binding of a standard molecule,may be ascertained. In addition, the absolute binding affinity can alsobe determined.

[0109] Determination of the nature ofthe interaction of a ligand with abiomolecular target may be carried out as exemplified for the binding ofa small molecule ligand with a nucleic acid target. A chimeric RNA/DNAbiomolecular target whose binding to a test ligand is to be studied isfirst prepared via automated synthesis protocols. An aliquot of a knownconcentration of chimeric nucleic acid is treated with a knownconcentration and quantity of a standard compound that is known to bindthat nucleic acid, such as the aminoglycoside paromomycin which is knownto bind to the 16S A-site of RNA. ESI-MS, followed by CID of theparomomycin-nucleic acid complex, affords a control spectrum for theinteractions and complex. A second aliquot of the chimeric nucleic acidis next treated with a test ligand using quantities and concentrationssimilar to those used for the control experiment. Application of themethod of the invention to this nucleic acid-ligand noncovalent complexaffords a test spectrum that reveals the nature of thebiomolecule-ligand interaction. Analysis of the noncovalent nucleicacid-ligand complex based on the known molecular weights of the twocomponents of the complex allows the determination of the number ofnucleic acid molecules and ligands present in the complex. Further,comparison of the abundance of the nucleic acid-ligand complex ion withthe abundance of the ion generated from the e.g. paromomycin-nucleicacid complex (or complex with any other known interacting species)provides a convenient and direct estimate of the binding affinity of thetest ligand compared to the standard, paromomycin. Since the standard iswell characterized, its solution binding affinity should be known fromother experiments or literature sources. For example, paromomycin bindsto a test 27-mer RNA with a ˜1 μM affinity. Knowing the binding affinityof the test ligand relative to paromomycin from the MS experiment, it isnow possible to determine the micromolar binding affinity of the testligand for the nucleic acid target being studied. Relative bindingaffinity may also be measured by testing a standard compound and testligand simultaneously as in a mixture with the target biomolecule, in asingle test assay.

[0110] Another object of the present invention is to provide generalmethods for the screening of compounds for drug discovery. The inventionprovides methods for the screening of a wide variety of biomoleculartargets that include, but are not limited to, peptides, proteins,receptors, antibodies, oligonucleotides, RNA, DNA, RNA/DNA hybrids,nucleic acids, oligosaccharides, carbohydrates, and glycopeptides. Themolecules that may be screened by using the methods of this inventioninclude, but are not limited to, organic or inorganic, small to largemolecular weight individual compounds, mixtures and combinatoriallibraries of ligands, inhibitors, agonists, antagonists, substrates, andbiopolymers, such as peptides or oligonucleotides.

[0111] The primary challenge when screening large collections andmixtures of compounds is not in finding biologically relevantactivities, for this has been demonstrated in many different cases, butin identifying the active components from such screens, and often frommixtures and pools of compounds that are found to be active. Onesolution that has been practiced by the art-skilled in high throughputdrug discovery is the iterative deconvolution of mixtures. Deconvolutionessentially entails the resynthesis of that combinatorial pool ormixture that was found to be active in screening against a target ofinterest. Resynthesis may result in the generation of a set of smallerpools or mixtures, or a set of individual compounds. Rescreening anditerative deconvolution are performed until the individual compoundsthat are responsible for the activity observed in the screens of theparent mixtures are isolated.

[0112] However, analytical techniques are limited in their ability toadequately handle the types of mixtures generated in combinatorialefforts. The similarity of members of combinatorial mixtures or pools,and the complexity of such mixtures, prohibit effective analyticalassessment until the mixtures have been deconvoluted into individualcompounds, or at the very least into pools of only a handful ofcomponents. While this process of deconvolution, involving resynthesis,rescreening and analysis, is very cumbersome and time-consuming, it isalso very costly. A general method that alleviates these problems byrapidly revealing active mixtures and identifying the active componentsof such mixtures is clearly needed to save time and money in the drugdiscovery process.

[0113] The present invention solves the need for a method to rapidlyassess the activity of combinatorial mixtures against a biomoleculartarget and also identify the structure of the active components of suchmixtures. This is exemplified by the screening of combinatorial mixturesfor binding to a nucleic acid target as follows. A chimeric RNA/DNAtarget of known sequence is selected as the screening target based onbiological relevance. This chimeric nucleic acid target is prepared viaautomated synthesis. An aliquot of the nucleic acid is used at aconcentration of 10 μM and treated with e.q. paromomycin acetate at aconcentration of 150 nM. A sample of the mixture is analyzed by themethod of the invention to demonstrate binding of the paromomycin byobservation of the paromomycin-nucleic acid complex ion. Next, analiquot of this mixture is treated with a DMSO solution of acombinatorial mixture of compounds such that the final concentration ofeach component of the mixture is ˜150 nM. This sample is then subjectedto ESI-MS, and the mass spectrum monitored for the appearance of newsignals that correspond to new nucleic acid-ligand noncovalent complexesformed with components of the combinatorial library.

[0114] The relative dissociation constants of these new complexes aredetermined by comparing the abundance of these new ions with theabundance of the paromomycin-nucleic acid complex ion whose bindingaffinity for the target is known a priori. Algorithmic deconvolution ofthe new complex ions observed, while taking into account the masses ofthe target and the components of the combinatorial library, provides themolecular weights of the binding ligands present in the observednoncovalent complexes. Alternatively, the identity of the binding ligandmay also be determined by first isolating the newly observed complex ionusing a triple quadrupole ion-trap or an ion cyclotron resonance device(ICR) followed by conventional identification by mass spectrometryfragment analysis. For example, upon isolation, a noncovalent complexion is ‘heated’ or dissociated into the constituent ligand andbiomolecule ions. This MS/MS experiment then can be tuned to studyfragmentation of the ligand. This information provides direct evidenceof the structure of the bound ligand. This method of the presentinvention, therefore, provides both the identity and relative bindingaffinity of members of combinatorial or other mixtures of compounds thatbind to the nucleic acid target.

[0115] Not only does the present invention provide methods for thedetermination of the molecular weight and absolute and relative bindingaffinity of the binding components of a combinatorial or other mixtureof compounds, but it also provides valuable information about the siteof binding on the biomolecular target. Such information permits theidentification of compounds having particular biological activity andgives rise to useful drugs, veterinary drugs, agricultural chemicals,industrial chemicals, diagnostics and other useful compounds. This canalso be accomplished as part of the same mass spectrometric procedure byisolating the newly observed complex ions using a triple quadrupoleion-trap or an ion cyclotron resonance device (ICR). For example, uponisolation, a noncovalent complex ion is collisionally activated tocleave the chimeric nucleic acid target at exposed deoxynucleotidesites. This MS/MS procedure, then, can be tuned to study fragmentationof the biomolecular target.

[0116] Comparison of the cleavage and fragment patterns so obtained forthe nucleic acid component of the noncovalent complex with patternsobtained for the native chimeric nucleic acid alone reveals thelocations on the nucleic acid that are protected by the binding of theligand. This indicates the binding sites for the ligand on the nucleicacid. Comparison of the cleavage patterns to those observed from the CIDof the standard-nucleic acid complex ion provide correlations betweenthe sites of binding of the new ligand and standard. In this fashion,ligands that bind to nucleic acid targets may be identified such thatthey compete for the same binding site on the nucleic acid where thestandard binds, or bind at completely different and new sites on thenucleic acid. Both these types of observations are of value from a drugdiscovery standpoint.

[0117] The methods of the present invention can be used to identifymetal ion binding sites on any of the biomolecules described herein.Preferably, the metal ion binding site binds alkali metals or alkalineearth metals. More preferably, the metal ions are Na⁺, Mg⁺⁺ and Mn⁺⁺.

[0118] Drug discovery, using any one of a number of different types ofbiomolecular targets attends use of the methods of this invention whichcan rapidly screen large combinatorial libraries and mixtures ofcompounds for binding activity against a specific target.

[0119] It is possible that combinatorial libraries and mixtures ofcompounds being used for screening may contain components that aresimilar in mass because their elemental compositions are similar whiletheir structures are different, or at the very least, isomeric orenantiomeric. In such instances, a simple algorithmic calculation of themolecular weight of a bound ligand will be insufficient to provide theidentity of the ligand for there may be multiple components of the samemolecular mass. The methods of the invention are also capable ofaddressing and resolving such problems of ligand identification. The useof MS/MS experiments to further fragment the bound ligand, followingselective ion accumulation of the ligand ion from the noncovalentcomplex, is a simple technique that provides structural detail of thebound ligand. This mass and structural information provided by themethods of this invention is expected to resolve the vast majority ofmass redundancy problems associated with the screening of largecombinatorial libraries and mixtures of compounds.

[0120] In a preferred embodiment, the present invention also providesmethod for simultaneously screening multiple biomolecular targetsagainst combinatorial libraries and mixtures or collections ofcompounds. This is a significant advantage of the present invention overcurrent state-of-the-art techniques in the screening of compounds forsuch binding. There is believed to be no prior technique that allows thesimultaneous and rapid screening of multiple targets, while providingstructural detail on the target and binding ligand at the same time. Inaddition to providing methods for the rapid and simultaneous screeningof multiple biomolecular targets, the present invention also providesmethods for determining the structure and nature of binding of both thetarget and binding ligand.

[0121] As discussed above, mass spectrometry methods of the presentinvention provide a direct means for screening and identifying thosecomponents of combinatorial mixtures that bind to a target biomoleculein solution. In order to enhance efficiency, it is preferable tomultiplex the screening process by simultaneously screening multipletargets for binding activity against a combinatorial library or mixtureof compounds. This strategy is normally limited by the distribution ofcharge states and the undesirable mass/charge overlap that will begenerated from all possible noncovalent biomolecule-ligand complexesthat could be formed during such a screening assay. This problem ofoverlapping peaks in the mass spectra is further exacerbated if thebiomolecular targets being screened are of similar sequence,composition, or molecular weight. In such instances it would not bepossible to ascertain in a rapid and simple operation the composition ofbiomolecule-ligand complexes because of the extensive mass redundancypresent in the pool of biomolecules being studied and possible in thecombinatorial library being screened.

[0122] The method of the present invention alleviates the problem ofbiomolecular target mass redundancy through the use of special massmodifying molecular weight tags. These mass modifying tags are typicallyuncharged or positively charged groups such as, but not limited to,alkyl and tetraalkylammonium groups, and polymers such as, but notlimited to, polyethylene glycols (PEG), polypropylene, polystyrene,cellulose, sephadex, dextrans, cyclodextrins, peptides, andpolyacrylamides. These mass modifying tags may be selected based ontheir molecular weight contribution and their ionic nature. These massmodifying tags may be attached to the biopolymeric targets at one ormore sites including, but not limited to, the 2′-O—, 3′-terminus,5′-terminus or along the sugar-phosphate backbone of nucleic acidtargets. Addition of mass modifying tags to the 5′terminus of syntheticoligonucleotides can be realized either using conventionalphosphoramidite chemistry, other conventional chemistry or bybiochemical or enzymatic means. Such mass modification of a nucleic acidmay be carried out using conventional, manual or automated techniques.Alternatively, addition of mass modifying tags may be performed at the3′-terminus by the use of appropriately modified polymer or CPG supportsfor solid-phase synthesis of nucleic acids. Mass modification at the3′terminus may also be done by biochemical or enzymatic means. It isalso possible to attach mass modifying tags to the internucleotidelinkages of a nucleic acid. This may be performed via the use ofappropriately modified phosphoramidites, or other nucleoside buildingblocks during nucleic acid synthesis or via post-synthetic modificationof the internucleotide linkage. Further, attachment of mass modifyingtags to nucleic acid targets may also be accomplished via the use ofbifunctional linkers at any functional site on the nucleic acid.Similarly, when working with other classes of biomolecular targets thesemass modifying tags may likewise be incorporated at one or morepositions on the biomolecule. As will be apparent, inclusion in eithertarget or ligand of isotopic mass labels may also be useful.

[0123] Thus, similar nucleic acid and other biological targets may bedifferentially tagged for rapid mass spectrometric screening by themethods of this invention. When noncovalent complexes are observed fromthis multiplexed screening ofmultiple nucleic acid targets with mixturesof small molecular weight combinatorial libraries, the constituentligand and biomolecule are readily identified using conventional massanalyzers such as quadrupole, ion trap, ICR, magnetic sector, or TOF andfollowed by MS/MS. This is because the mass modifying tags make the m/z(mass to charge ratio) of the signal arising from each targetbiomolecule-ligand complex ion of similar charge, distinct in the massspectrum, and which results in cleanly separated ion peaks. Massredundancy and peak overlap are both avoided by the use of massmodifying tags.

[0124] The present invention is also highly useful in combination withother techniques for the identification of ligands which interact withmolecular interaction sites on RNA and other nucleic acids. Molecularinteraction sites attend RNA and are believe to be highly important inthe functioning of such RNA. The nucleotide sequences of molecularinteraction sites are highly conserved, even among taxonomically diversespecies. Moreover, such molecular interaction sites have specificstructures which provide opportunities for ligand binding. Ascertainingwhich ligands bind to such sites as well as determining the relativeaffinities and specificities for the binding of each ligand provideslead compounds for drug discovery, therapeutics, agricultural chemistry,industrial chemistry and otherwise.

[0125] The present mass spectrometric techniques, especially the MASStechniques and those which possess similar analytical robustness andpower, are ideally suited for cooperating with drug and other discoveryand identification programs such as those which determine ligand bindingto molecular interaction sites. The identification of molecularinteraction sites in RNA and other nucleic acids and the determinationof hierarchies of molecular ligands which likely bind to such molecularinteraction sites can be evaluated through the present techniques. Thus,in accordance with preferred embodiments of the present invention, ahierarchy of ligands ranked in accordance with their anticipated orcalculated likelihood of binding to a molecular interaction site of anRNA are actually synthesized. Such synthesis is preferably accomplishedin an automated or robotized fashion, preferably from instruction setsprovided in attendance to the ranked hierarchy of ligands. The compoundsmay be prepared in a library or mixture since the present massspectrometric methods can evaluate pluralities of compounds and theircomplexes with RNA simultaneously.

[0126] After the ligands are synthesized, preferably in library form,they are contacted with the RNA having the molecular interaction site ofinterest. Complexation or binding (conventionally, non-covalent binding)is permitted to occur. The complexed RNA—ligand library is then analyzedby mass spectrometry. A principal object of the analysis is preferablythe determination of which ligands bind to the RNA molecular interactionsite and, among those, which ones rank more highly in terms ofspecificity and affinity. Accordingly, it is possible to identify from amixture or library of compounds, which ones are the most interactivewith a particular molecular interaction site so as to be able tomodulate it. Such compounds can either be used themselves, or, morelikely, be used as lead compounds for modification into drugs,agricultural chemicals, environmental chemicals, industrial and foodchemicals and otherwise.

[0127] As described above, it is highly desirable to challenge RNAshaving molecular interaction sites with libraries of compounds whichhave already been predicted or calculated to be likely to interact withthe interaction sites. It is preferred that such molecules belong toranked hierarchies so as to give rise to the greatest likelihood offinding highly potent modulators of the target RNA.

[0128] While there are a number of ways to identify compounds likely tointeract with molecular interaction sites of RNA and other biologicalmolecules, preferred methodologies are described in U.S. patentapplications filed on even date herewith and assigned to the assignee ofthis invention. These application bear U.S. Serial Nos. (Unknown) andhave been assigned attorney docket numbers IBIS-0002, IBIS 0003,IBIS-0004, IBIS-0006 and IBIS-0007. All of the foregoing applicationsare incorporated by reference herein in their entirety.

[0129] One mass spectrometric method which is particularly useful whencombined with the techniques of the foregoing commonly owned inventionsprovides the determination of specificity and affinity of ligands to RNAtargets. MASS (multi target affinity/specificity screening) techniquescan provide high throughput screening methods to analyze the specificityand affinity of ligands to molecular interaction sites of nucleic acids,especially RNA. MASS employs high performance electrospray ionizationFourier transform ion cyclotron resonance mass spectrometry(ESI-FTICR-MS) to a) determine exact chemical composition of affinityselected ligands originating from a combinatorial library, b) determinerelative dissociation constants (Kd) of ligands complexed to thetarget(s), and c) determine the location of ligand binding. Thisinformation can be gathered from each target(s) or library set in asingle assay in less than 15 minutes. This scheme benefits from twounique aspects of the ESI-FTICR combination. The “soft” nature of theelectrospray ionization process allows specific noncovalent complexes tobe ionized and transferred into the gas phase intact where they areamenable to subsequent characterization by mass spectrometry. The highresolving power afforded by the FTICR platform facilitates thecharacterization of complex mixtures which, when combined with the highmass accuracy inherent to FTICR, provides unambiguous identification ofligands complexed with the molecular interaction site or sites of atarget or targets.

[0130] Binding site information can be obtained by comparing the gasphase fragmentation patterns of the free and complexed target andabsolute binding affinities while relative binding constants are derivedfrom the relative abundance of complexes using a complex with a known Kdas an internal standard. With knowledge of the specificity and affinityof ligands to the molecular interaction site of a target RNA, thedesired lead or ultimate compound for modulation of the RNA can bedetermined. Therapeutic, agricultural chemical, industrial chemical andother products which benefit from modulation of such RNA attend thisresult.

[0131] The simultaneous screening of a combinatorial library ofmolecules of mass 700-750, against two nucleic acid targets of the samemolecular weight but different sequence, is demonstrated by the use ofmass modifying tags. If both nucleic acids targets being studied are27-mer RNAs of mass 8927, then screening a library of molecules of mass700-750 could afford a bewildering jumble of noncovalent complex ions inthe mass spectrum of the mixture of the two nucleic acids and thelibrary. However if one of the two targets is mass modified, for exampleby the use of a PEG chain of mass 3575 attached at the 5′ terminus ofthe target, then the mass spectrum will be significantly simplified. Itis known that a 27-mer will generate multiply-charged ion signals,following electrospray ionization, of mass/charge values 1486.8, 1784.4,and 2230.8 for the (M-6H)⁶⁻, (M-5H)⁵⁻, and the (M-4H)⁴⁻ charge states.Upon binding to small molecules of mass 700-750, the unmodifiedRNA-ligand complexes are anticipated to occur in the 1603.2-1611.6,1924.4-1934.4, and 2405.8-2418.3 m/z range. If the second nucleic acidtarget were not modified in any way, the signals from its complexeswould have occurred in the same regions. However, using the massmodified RNA, bearing the PEG chain of mass 3575, results in theobservation of the corresponding mass modified RNA-ligand complexes tooccur in the 2199-2207.4, 2639-2649 and 3299-3311 m/z range. Thus allsignals from the second mass modified nucleic acid would be cleanlyresolved from those of the first RNA. These noncovalent complex ions maybe selected e.g. by triple, quadrupole, ion trap or ICR techniques, andstudied further by MS/MS to afford detailed understanding of the sitesof ligand-RNA interaction, and the nature of these interactions, as hasbeen discussed above.

[0132] In a further embodiment, the methods of this invention areapplicable for the determination of the specificity of bindinginteractions between a ligand and a biomolecular target. Bysimultaneously screening multiple biomolecular targets with one or morecompounds, using the methods of this invention, it is possible toascertain whether a ligand binds specifically to only one targetbiomolecule, or whether the binding observed with the target isreproduced with control biomolecules as well, and is thereforenon-specific. This is an important distinction to be made when screeninglarge libraries and collections of compounds for binding to biomoleculartargets. It is desirable to quickly distinguish those ligands that areselective or specific for the biomolecular target of interest from thosethat are non-specific and bind to any and all targets. From thestandpoint of drug discovery, it is most often the case that undesirablebiological activities arise from the indiscriminate, non-specificbinding of molecules to unrelated biomolecules. The present inventionprovides a valuable and straightforward method for assessing thespecificity of interactions between a ligand and a panel of targets.

[0133] The use of mass modifying tags for the simultaneous screening ofmultiple biomolecular targets is applicable to the determination ofbinding specificity of a ligand as well. Mass modifying tags may be usedto differentiate several biomolecular targets that serve as a controlpanel for screening a combinatorial library of individual compoundsagainst a specific biomolecular target. When simultaneously screeningmultiple biomolecular targets using the mass spectrometric methods ofthis invention, it is necessary to ensure good separation of the ionsthat result from each target and its complex with the binding ligand.This peak overlap is easily eliminated by the facile introduction ofdifferent mass modifying tags onto the different biomblecular targetsbeing studied. A mixture of the biomolecular target and the controlpanel is mixed with the ligand being evaluated. This solution is thenionized by ESI-MS, and the noncovalent complex ions observed may bedirectly identified as having resulted from the binding of the ligand toa specific target from the several biomolecular targets present in themixture. In this way, a qualitative indication of specificity orselectivity of binding for the desired target versus the controlbiomolecules may be obtained. This selectivity may also be quantitatedthrough the use of appropriate standards of known binding affinity andcomparison of the ligand-biomolecule complex ion abundance to theabundance of the standard-biomolecule abundance. Further, details on thenature of the specific or non-specific interaction of the ligand withthe different biomolecules may also be obtained following ion-selectionand subsequent MS/MS experiments, as discussed above.

[0134] Likewise, it is also possible to determine the proportionalbinding of a ligand to two or more biomolecular targets using themethods of this invention. Thus by the use-of appropriate mass modifyingtags on the different biomolecular targets, the different noncovalentcomplexes formed via differential binding of the ligand can be readilydistinguished in the mass spectrometer. Quantitation of the binding ispossible by measuring the abundance of these ions. Comparing therelative abundances of these ions provides a means to determine theproportional binding of the ligand to the different biomoleculartargets.

[0135] Yet another application of the methods of the present inventionis to determine the differential binding of ligands to biomoleculartargets of different origin. When studying the binding of small moleculeligands to RNA targets, it is straightforward to distinguish between thenoncovalent ligand-RNA complexes generated from binding to the twodifferent RNA targets, even though both may be screened simultaneouslyas a mixture in the same assay. Further, it is also possible todetermine specificity and selectivity of the ligand for one versus theother RNA, and to determine the relative affinities of binding to eachRNA target.

[0136] The methods of the present invention are applicable to the studyof a wide variety of biomolecular targets that include, but are notlimited to, peptides, proteins, receptors, antibodies, oligonucleotides,RNA, DNA, RNA/DNA hybrids, nucleic acids, modified oligonucleotides,peptide-nucleic acids (PNAs), oligosaccharides, carbohydrates, andglycopeptides. Further these biomolecular targets may be synthetic orisolated from natural sources. Biomolecular targets of natural origininclude, but are not limited to, those obtained from microbial, plant,animal, viral or human materials, such as, but not limited to, cells,cell extracts, fluids, tissues and organs.

[0137] The molecules that may be screened by using the methods of thisinvention include, but are not limited to, organic or inorganic, smallto large molecular weight individual compounds, and combinatorialmixture or libraries of ligands, inhibitors, agonists, antagonists,substrates, and biopolymers, such as peptides or oligonucleotides.

[0138] Combinatorial mixtures include, but are not limited to,collections of compounds, and libraries of compounds. These mixtures maybe generated via combinatorial synthesis of mixtures or via admixture ofindividual compounds. Collections of compounds include, but are notlimited to, sets of individual compounds or sets of mixtures or pools ofcompounds. These combinatorial libraries may be obtained from syntheticor from natural sources such as, for example to, microbial, plant,marine, viral and animal materials. Combinatorial libraries include atleast about twenty compounds and as many as a thousands of individualcompounds and potentially even more. When combinatorial libraries aremixtures of compounds these mixtures typically contain from 20 to 5000compounds preferably from 50-1000, more preferably from 50-100.Combinations of from 100-500 are useful as are mixtures having from500-1000 individual species. Typically, members of combinatoriallibraries have molecular weight less than about 5000 Da.

[0139] The mass spectrometry techniques that may be used in the methodsof this invention include all of the techniques and systems describedherein or are subsequently developed. Tandem techniques are also useful,including combinations of all of the foregoing and LC/MS. The massspectrometers used in the methods of this invention may be a singlequadrupole, triple quadrupole, magnetic sector, quadrupole ion trap,time-of-flight instrument, and FTICR. Future modifications to massspectrometry are expected to give rise to improved techniques which mayalso be useful herein.

[0140] In another embodiment of the present invention, binding ofmixtures of aminoglycosides can be measured simultaneously againstmultiple RNA targets of identical length and similar (or identical)molecular weight. Addition of a neutral mass tag to one of the RNAtargets shifts those to a higher mass/charge ratio, where complexes withsmall molecules can be identified unambiguously. An appropriately placedneutral mass tag does not alter RNA-ligand binding. Preferably, thismethod is demonstrated with model RNAs corresponding to the decodingregion ofthe prokaryotic and eukaryotic small subunit rRNAs and amixture of compounds, such as, for example, five aminoglycosides. In theexamples set forth below, complexes are observed between theaminoglycoside library and the prokaryotic rRNA model, while noaminoglycoside was observed to bind to the mass tagged eukaryotic rRNAmodel. The differential binding data is consistent with the eukaryoticA-site rRNA having a different confirmation compared to the prokaryoticA-site that prevents entry and binding of neomycin-classaminoglycosides. Mass spectrometric analysis of neutral mass-taggedmacromolecular targets represents a new high throughput screeningparadigm in which the interaction of multiple targets against acollection of small molecules can be evaluated in parallel.

[0141] The preferred model system employed herein comprises a librarycomprised of five 2-deoxystreptamine aminoglycoside antibiotics whichhave a range of binding affinities for the decoding sites of theprokaryotic and eukaryotic ribosomal RNA ranging from ˜28 nM to ˜1.5 mM.FIG. 24 illustrates the secondary structures for the 27-nucleotidemodels of the 16S and 18S rRNA decoding sites. These constructs consistof a 7 base pair stem structure containing a non-canonical U-U and apurine-adenosine mismatch base pair adjacent to a bulged adenosineresidue closed by a UUCG tetraloop. NMR studies of a complex between 16Sand paromomycin show that the RNA makes primary hydrogen bond,electrostatic, and stacking contacts with the aminoglycoside (Fourmy, etal., Science, 1996, 274, 1367-1371) and that paromomycin binds in themajor groove of the model A-site RNA within the pocket created by theA-A base pair and the single bulged adenine. The masses for the two RNAmodels differ by only 15.011 Da and the (M-5H⁺)⁵⁻ species of theseconstructs differ by only 3 m/z units. While the high resolutioncapabilities of the FTICR mass spectrometer can easily resolve thesespecies, mass spectra from a solution containing both RNAs arecomplicated by overlap among the signals from free RNA ions and theirsodium and potassium-adducted species.

[0142] Methods to increase the separation between the associated signalsin the mass spectra due to overlap among signals from RNAs 16S and 18Sare described herein. RNA targets modified with additional unchargedfunctional groups conjugated to their 5′-termini have been synthesized.Such a synthetic modification is referred to herein as a neutral masstag. The shift in mass, and concomitant m/z of a mass-taggedmacromolecule moves the family of signals produced by the tagged RNAinto a resolved region of the mass spectrum.

[0143] When simultaneously screening of untagged 16S and untagged 18Sagainst a combinatorial library of small molecules, if a complex wereobserved at 515.011 Da higher than 16S, it would not be possible todirectly determine (without tandem MS methods) whether the complexcorresponded to a ligand weighing 515.011 Da complexed to the 16S targetor a ligand weighing 500.000 Daltons complexed to 185. Furthermore,because positively charged ligands can have non-specific interactionswith RNA oligomers, it is often desirable to assay libraries forspecific and non-specific binding by screening against two or more RNAtargets simultaneously (e.g. a structured target sequence and anunstructured control sequence) in a single ESI-MS experiment. Thismultiplex advantage can be further exploited in the RNA-drug discoveryarena in which libraries are to be assayed against multiple RNA targetsof similar, or identical, mass. A single analysis in which 5 RNA targetsare screened against a combinatorial library of 200 componentsfacilitates the direct evaluation of 1000 RNA-ligand interactions fromthe acquisition of a single mass spectrum.

[0144] While the ability to shift the m/z range of closely relatedmacromolecules is highly desirable as described above, it is preferablydesired that the mass tag does not alter key physical properties of thetarget or the ligand binding properties. Preferably, an 18-atom mass tag(C₁₂H₂₅O₉) attached to the 5′-terminus of the RNA oligomer through aphosphodiester linkage can be employed. This mass tag has no appreciableaffect on oligonucleotide solubility, ionization efficiency, or UVabsorbance, and does not alter RNA-ligand binding. This latter attributeis evidenced by the data in FIG. 25 that illustrates the conserved ratioof free:bound RNA for the untagged and tagged RNA models of thebacterial decoding site under competitive binding conditions withparomomycin.

[0145] Aminoglycoside antibiotics inhibit bacterial growth by disruptingessential prokaryotic RNA-protein and RNA-RNA interactions. In vivo,atherapeutic effect is realized because paromomycin alters essential RNAinteractions in prokaryotes (by binding to the 16S A-site with highaffinity) but does appreciably disrupt the function of the eukaryoticRNA complexes (owing to the low affinity of paromomycin for the 18SA-site). A compound that binds both the 16S and 18S A-sites with similaraffinity would likely inhibit bacterial growth but might also havedeleterious cytotoxic effects in eukaryotic cells and would not make asuitable therapeutic agent. Thus, the 16S/18S model RNA system can servenot only as an interesting target for new generation antibiotics, but asa well characterized control for our mass spectrometry based RNA-ligandaffinity assay.

[0146] The ESI-FTICR mass spectrum depicted in FIG. 26 was acquired froma 10 mM mixture of untagged 16S and tagged 18S in the presence of anequimolar mixture of five aminoglycosides. It is to be understood thatother biomolecules may be used in place of the aminoglycosides. Theaminoglycosides have been selected from two classes of2-deoxystreptamines: 4,5-disubstituted (paromomycin, and lividomycin),and 4,6-disubstituted (tobramycin, sisomicin, and bekanamycin), presentat 500 nM each. Complexes corresponding to 1:1 binding of individualaminoglycosides were observed between 16S and all members of theaminoglycoside mixture, with the apparent affinities estimated from theabundances of the respective complexes differing substantially. Signalintensities from the complexes with paromomycin (m/z 1925.572) andlividomycin (m/z 1954.790) are consistent with MS-measured dissociationconstants of 110 nM and 28 nM, respectively. The intensities of 16Scomplexes with tobramycin (m/z 1895.960), bekanamycin (m/z 1899.171),and sisomicin (m/z 1891.972) were reduced, consistent with solutiondissociation constants of 1.5 mM. Wang, et al., Biochemistry, 1997, 36,768-779. Hence, under these assay conditions, the MS-observed ionabundances reflect the solution dissociation constants. The inset inFIG. 26 demonstrates the ability to resolve the isotopic envelope foreach complex and allows mass differences to be calculated fromhomo-isotopic species, thus, measuring the difference in m/z between theRNA target and the RNA-ligand complex allows precise mass determinationof the ligand. The spectrum is calibrated using multiple isotope peaksof the (M-5H⁺)⁵⁻ and (M-4H⁺)⁴⁻ charge states of the free RNA as internalmass standards which brackets the m/z range in which complexes areobserved. The average mass measurement error obtained for the complexesin FIG. 26 is 2.1 ppm when m/z differences are measured between the mostabundant (4 ¹³C) isotope peak of 16S and each complex. This postcalibration scheme is easily automated which enables rapid, highprecision mass measurements of affinity selected ligands againstmultiple targets in a high throughput mode.

[0147] The enhanced affinity of lividomycin for 16S relative to theaffinity of paromomycin for 16S is interesting. While lividomycin isbelieved to bind to the 16S ribosomal subunit, the exact site ofinteraction has not been established. Lividomycin has two significantstructural differences from paromomycin. First, the additionalmannopyranosyl ring could generate new macromolecular contacts with theRNA. However, the orientation of paromomycin ring IV is disordered inthe NMR-derived structure for the complex with 16S. In addition, ahydroxyl group on ring I that makes a contact with A1492 is missing. Therelatively high abundance of the 16S-lividomycin complex suggests thatlividomycin binds at or near the 16S A-site, and generates additionalcontacts that enhance the binding affinity nearly 4-fold. Perhaps themost striking feature of the spectrum in FIG. 26 is the complete absenceof complexes between 18S and paromomycin or lividomycin. This resultsuggests there must be poor shape and electrostatic complementaritybetween the 4,5-disubstituted 2-DOS class of aminoglycoside and theconserved architecture of the eukaryotic ribosomal decoding site.

[0148] Thus, according to the invention, RNA targets with similar (oridentical) molecular masses can be labeled with small neutral moleculesto measure binding between the targets and ligands using massspectrometry. By screening multiple targets against ligand mixturessimultaneously, the information content of the assay is enhanced,resulting in a dramatic reduction in the number of analyses required.Although the increased complexity of the multi-substrate/ligand mixturesplaces high demands on the mass analyzer, the methods described hereinfacilitate the simultaneous analysis of numerous targets under identicalsolution conditions and ligand concentrations, further enhancing thehigh-throughput nature of the screening strategy and allowing directcomparisons of binding affinities for closely related targets. Thisconcept of “rational” target design should also be applicable in studiesof proteins that differ in amino acid sequence.

EXAMPLES Example 1 Determining the Structure of a 27-mer RNAcorresponding to the 16S rRNA A site

[0149] In order to study the structure of the 27-mer RNA correspondingto the 16S rRNA A site, of sequence5′-GGC-GUC-ACA-CCU-UCG-GGU-GAA-GUC-GCC-3′ (SEQ ID NO:1) a chimericRNA/DNA molecule that incorporates three deoxyadenosine (dA) residues atpositions 7, 20 and 21 was prepared using standard nucleic acidsynthesis protocols on an automated synthesizer. This chimeric nucleicacid of sequence 5′-GGC-GUC-dACA-CCU-UCG-GGU-GdAdA-GUC-GCC-3′ (SEQ IDNO:2) was injected as a solution in water into an electrospray massspectrometer. Electrospray ionization of the chimeric afforded a set ofmultiply charged ions from which the ion corresponding to the (M-5H)⁵⁻form of the nucleic acid was further studied by subjecting it tocollisionally induced dissociation (CID). The ion was found to becleaved by the CID to afford three fragments of m/z 1006.1, 1162.8 and1066.2. These fragments correspond to the w₇ ⁽²⁻⁾, w₈ ⁽²⁻⁾ and thea₇-B⁽²⁻⁾ fragments respectively, that are formed by cleavage of thechimeric nucleic acid adjacent to each of the incorporated dA residues.

[0150] The observation that cleavage and fragmentation of the chimericRNA/DNA has occurred adjacent to all three dA sites indicates that thetest RNA is not ordered around the locations where the dA residues wereincorporated. Therefore, the test RNA is not structured at the 7, 20 and21 positions.

[0151] A systematic series of chimeric RNA/DNA molecules is synthesizedsuch that a variety of molecules, each incorporating deoxy residues atdifferent site(s) in the RNA. All such RNA/DNA members are comixed intoone solution. MS analysis, as described above, are conducted on thecomixture to provide a complete map or ‘footprint’ that indicates theresidues that are involved in secondary or tertiary structure and thoseresidues that are not involved in any structure. See FIG. 1.

Example 2 Determining the Binding Site for Paromomycin on a 27-mer RNAcorresponding to the 16S rRNA A site

[0152] In order to study the binding of paromomycin to the RNA ofexample 1, the chimeric RNA/DNA molecule of example 1 was synthesizedusing standard automated nucleic acid synthesis protocols on anautomated synthesizer. A sample of this nucleic acid was then subjectedto ESI followed by CID in a mass spectrometer to afford thefragmentation pattern indicating a lack of structure at the sites of dAincorporation, as described in Example 1. This indicated theaccessibility of these dA sites in the structure of the chimeric nucleicacid.

[0153] Next, another sample of the chimeric nucleic acid was treatedwith a solution of paromomycin and the resulting mixture analyzed by ESIfollowed by CID using a mass spectrometer. The electrospray ionizationwas found to produce a set of multiply charged ions that was differentfrom that observed for the nucleic acid alone. This was also indicativeof binding of the paromomycin to the chimeric nucleic acid, because ofthe increased mass of the observed ion complex. Further, there was alsoobserved, a shift in the distribution of the multiply charged ioncomplexes which reflected a change in the conformation of the nucleicacid in the paromomycin-nucleic acid complex into a more compactstructure.

[0154] Cleavage and fragmentation of the complex by CID affordedinformation regarding the location of binding of the paromomycin to thechimeric nucleic acid. CD was found to produce no fragmentation at thedA sites in the nucleic acid. Thus paromomycin must bind at or near allthree dA residues. Paromomycin therefore is believed to bind to the dAbulge in this RNA/DNA chimeric target, and induces a conformationalchange that protects all three dA residues from being cleaved duringmass spectrometry. See FIG. 2.

Example 3 Determining the Identity of Members of a Combinatorial Librarythat Bind to a Biomolecular Target

[0155] 1 mL (0.6 O.D.) of a solution of a 27-mer RNA containing 3 dAresidues (from Example 1) was diluted into 500 μL of 1:1isopropanol:water and adjusted to provide a solution that was 150 mM inammonium acetate, pH 7.4 and wherein the RNA concentration was 10 mM. Tothis solution was added an aliquot of a solution of paromomycin acetateto a concentration of 150 nM. This mixture was then subjected to ESI-MSand the ionization of the nucleic acid and its complex monitored in themass spectrum. A peak corresponding to the (M-5H)⁵⁻ ion of theparomomycin-27mer complex is observed at an m/z value of 1907.6. Asexpected, excess 27-mer is also observed in the mass spectrum as its(M-5H)⁵⁻ peak at about 1784. The mass spectrum confirms the formation ofonly a 1:1 complex at 1907.6 (as would be expected from the addition ofthe masses of the 27-mer and paromomycin) and the absence of any biscomplex that would be expected to appear at an m/z of 2036.5.

[0156] To the mixture of the 27-mer RNA/DNA chimeric and paromomycin wasnext added 0.7 mL of a 10 μM stock solution of a combinatorial librarysuch that the final concentration of each member of the combinatoriallibrary in this mixture with 27-mer target was ˜150 nM. This mixture ofthe 27-mer, paromomycin and combinatorial compounds was next infusedinto an ESI-MS at a rate of 5 mL/min. and a total of 50 scans weresummed (4 microscans each), with 2 minutes of signal averaging, toafford the mass spectrum of the mixture.

[0157] The ESI mass spectrum so obtained, shown in FIG. 3, demonstratedthe presence of new signals for the (M-5H)⁵⁻ ions at m/z values of1897.8, 1891.3 and 1884.4. Comparing these new signals to the ion peakfor the 27-mer alone the observed values of m/z of those members of thecombinatorial library that are binding to the target can be calculated.The masses of the binding members of the library were determined to be566.5, 534.5 and 482.5, respectively. Knowing the structure of thescaffold, and substituents used in the generation of this library, itwas possible to determine what substitution pattern (combination ofsubstituents) was present in the binding molecules.

[0158] It was determined that the species of m/z 482.5, 534.5 and 566.5would be the library members that bore the acetic acid +MPAC groups, thearomatic +piperidyl guanidine groups and the MPAC +guanidylethylamidegroups, respectively. In this manner, if the composition of thecombinatorial library is known a priori, then the identity of thebinding components is straightforward to elucidate.

[0159] The use of FTMS instrumentation in such a procedure enhances boththe sensitivity and the accuracy of the method. With FTMS, this methodis able to significantly decrease the chemical noise observed during theelectro spray mass spectrometry of these samples, thereby facilitatingthe detection of more binders that may be much weaker in their bindingaffinity. Further, using FTMS, the high resolution of the instrumentprovides accurate assessment of the mass of binding components of thecombinatorial library and therefore direct determination of the identityof these components if the structural make up of the library is known.

Example 4 Determining the Site of Binding for Members of a CombinatorialLibrary that Bind to a Biomolecular Target

[0160] The mixture of 27-mer RNA/DNA chimeric nucleic acid, as target,with paromomycin and the combinatorial library of compounds from Example3 was subjected to the same ESI-MS method as described in Example 3. TheESI spectrum from Example 2 showed new signals arising from thecomplexes formed from binding of library members to the target, at m/zvalues of 1897.8,1891.3 and 1884.4. The paromomycin-27mer complex ionwas observed at an m/z of 1907.3.

[0161] Two complex ions were selected from this spectrum for furtherresolution to determine the site of binding of their component ligandson the 27-mer RNA/DNA chimeric. First, the ions at 1907.3, thatcorrespond to the paromomycin-27mer complex, were isolated via anion-isolation procedure and then subjected to CID. No cleavage was foundto occur and no fragmentation was observed in the mass spectrum. Thisindicates that the paromomycin binds at or near in the bulged region ofthis nucleic acid where the three dA residues are present. Paromomycintherefore protects the dA residues in the complex from fragmentation byCID.

[0162] Similarly, the ions at m/z 1897.8, that correspond to the complexof a library member with the 27mer target, were isolated via anion-isolation procedure and then subjected to CID using the sameconditions used for the previous complex, and the data was averaged for3 minutes. The resulting mass spectrum (FIG. 4) revealed six majorfragment ions at M/z values of 1005.8, 1065.6, 1162.8, 2341.1, 2406.3and 2446.0. The three fragments at m/z 1005.8, 1065.6 and 1162.8correspond to the w₆ ⁽²⁻⁾, a₇-B⁽²⁻⁾ and w₇ ⁽²⁻⁾ ions from the nucleicacid target. The three ions at higher masses of 2341.1, 2406.3 and2446.0 correspond to the a₂₀-B⁽³⁻⁾ ion+566 Da, w₂₁ ⁽³⁻⁾ ion+566 Da andthe a₂₁-B⁽³⁻⁾ ion+566 Da. The data demonstrates at least two findings:first, since only the nucleic acid can be activated to give fragmentions in this ESI-CID experiment, the observation of new fragment ionsindicates that the 1897.8 ion peak results from a library member boundto the nucleic acid target. Second, the library member has a molecularweight of 566. This library member binds to the GCUU tetraloop or thefour base pairs in the stem structure of the nucleic acid target (theRNA/DNA. chimeric corresponding to the 16S rRNA A site) and it does notbind to the bulged A site or the 6-base pair stem that contains the U*Umismatch pair of the nucleic acid target.

[0163] Further detail on the binding site of the library member can begained by studying its interaction with and influence on fragmentationoftarget nucleic acid molecules where the positions of deoxynucleotideincorporation are different.

Example 5 Determining the Identity of a Member of a CombinatorialLibrary that Binds to a Biomolecular Target and the Location of Bindingto the Target

[0164] A 10 mM solution of the 27-mer RNA target, corresponding to the16S rRNA A-site that contains 3 dA residues (from Example 1), in 100 mMammonium acetate at pH 7.4 was treated with a solution of paromomycinacetate and an aliquot of a DMSO solution of a second combinatoriallibrary to be screened. The amount of paromomycin added was adjusted toafford a final concentration of 150 nM. Likewise, the amount of DMSOsolution of the library that was added was adjusted so that the finalconcentration of each of the 216 member components of the library was˜150 nM. The solution was infused into a Finnigan LCQ ion trap massspectrometer and ionized by electrospray. A range of 1000-3000 m/z wasscanned for ions of the nucleic acid target and its complexes generatedfrom binding with paromomycin and members of the combinatorial library.Typically 200 scans were averaged for 5 minutes. The ions from thenucleic acid target were observed at m/z 1784.4 for the (M-5H)⁵⁻ ion and2230.8 for the (M-4H)⁴⁻ ion. The paromomycin-nucleic acid complex wasalso observed as signals of the (M-5H)⁵⁻ ion at m/z 1907.1 and the(M-4H)⁴⁻ ion at m/z 2384.4 u.

[0165] Analysis of the spectrum for complexes of members of thecombinatorial library and the nucleic acid target revealed several newsignals that arise from the noncovalent binding of members of thelibrary with the nucleic acid target. At least six signals for suchnoncovalent complexes were observed in the mass spectrum. Of these thesignal at the lowest m/z value was found to be a very strong binder tothe nucleic acid target. Comparison of the abundance of thisligand-nucleic acid complex ion with the abundance of the ion derivedfrom the paromomycin-nucleic acid complex revealed a relative bindingaffinity (apparent K_(D)) that was similar to that for paromomycin.

[0166] MS/MS experiments, with ˜6 minutes of signal averaging, were alsoperformed on this complex to further establish the molecular weight ofthe bound ligand. A mass of 730.0±2 Da was determined, since theinstrument performance was accurate only to ±1.5 Da. Based on thisobserved mass of the bound ligand and the known structures of thescaffold and substituents used in generating the combinatorial library,the structure of the ligand was determined to bear either of threepossible combinations of substituents on the PAP5 scaffold. The MS/MSanalysis of this complex also revealed weak protection of the dAresidues of the hybrid RNA/DNA from CID cleavage. Observation offragments with mass increases of 730 Da showed that the molecule bindsto the upper stem-loop region of the rRNA target.

Example 6 Determining the Identity of Members of a Combinatorial Librarythat Bind to a Biomolecular Target and the Location of Binding to theTarget

[0167] A 10 mM solution of the 27-mer RNA target, corresponding to the16S rRNA A-site that contains 3 dA residues (from Example 1), in 100 mMammonium acetate at pH 7.4 was treated with a solution of paromomycinacetate and an aliquot of a DMSO solution of a third combinatoriallibrary to be screened. The amount of paromomycin added was adjusted toafford a final concentration of 150 nM. Likewise, the amount of DMSOsolution of the library that was added was adjusted so that the finalconcentration of each of the 216 member components of the library was˜150 nM. The solution was infused into a Finnigan LCQ ion trap massspectrometer and ionized by electrospray. A range of 1000-3000 m/z wasscanned for ions of the nucleic acid target and its complexes generatedfrom binding with paromomycin and members of the combinatorial library.Typically 200 scans were averaged for 5 minutes. The ions from thenucleic acid target were observed at m/z 1784.4 for the (M-5)⁵⁻ ion and2230.8 for the (M-4H)⁴⁻ ion. The paromomycin-nucleic acid complex wasalso observed as signals of the (M-5H)⁵⁻ ion at m/z 1907.1 and the(M-4H)⁴⁻ ion at m/z 2384.4 u.

[0168] Analysis of the spectrum for complexes of members of thecombinatorial library and the nucleic acid target revealed several newsignals that arise from the noncovalent binding of members of thelibrary with the nucleic acid target. At least two major signals forsuch noncovalent complexes were observed in the mass spectrum. MS/MSexperiments, with ˜6 minutes of signal averaging, were also performed onthese two complexes to further establish the molecular weights of thebound ligands.

[0169] The first complex was found to arise from the binding of amolecule of mass 720.2±2 Da to the target. Two possible structures werededuced for this member of the combinatorial library based on thestructure of the scaffold and substituents used to build the library.These include a structure of mass 720.4 and a structure of mass 721.1.MS/MS experiments on this ligand-target complex ion using CIDdemonstrated strong protection of the A residues in the bulge structureof the target. Therefore this ligand must bind strongly to the bulged dAresidues of the RNA/DNA target.

[0170] The second major complex observed from the screening of thislibrary was found to arise from the binding of a molecule of mass665.2±2 Da to the target. Two possible structures were deduced for thismember of the library based on the structure of the scaffold andsubstituents used to build the library. MS/MS experiments on thisligand-target complex ion using CID demonstrated strong fragmentation ofthe target. Therefore this ligand must not bind strongly to the bulgeddA residues of the RNA/DNA target. Instead the fragmentation pattern,together with the observation of added mass bound to fragments from theloop portion of the target, suggest See FIG. 6. that this ligand mustbind to residues in the loop region of the RNA/DNA target.

Example 7 Simultaneous Screening of a Combinatorial Library of Compoundsagainst Two Nucleic Acid Targets

[0171] The two RNA targets to be screened are synthesized usingautomated nucleic acid synthesizers. The first target (A) is the 27-merRNA corresponding to the 16S rRNA A site and contains 3 dA residues, asin Example 1. The second target (B) is the 27-mer RNA bearing 3 dAresidues, and is of identical base composition but completely scrambledsequence compared to target (A). Target (B) is modified in the last stepof automated synthesis by the addition of a mass modifying tag, apolyethylene glycol (PEG) phosphoramidite to its 5′-terminus. Thisresults in a mass increment of 3575 in target (B), which bears a massmodifying tag, compared to target (A).

[0172] A solution containing 10 mM target (A) and 10 mM mass modifiedtarget (B) is prepared by dissolving appropriate amounts of both targetsinto 100 mM ammonium acetate at pH 7.4. This solution is treated with asolution of paromomycin acetate and an aliquot of a DMSO solution of thecombinatorial library to be screened. The amount of paromomycin added isadjusted to afford a final concentration of 150 nM. Likewise, the amountof DMSO solution of the library that is added is adjusted so that thefinal concentration of each of the 216 member components of the libraryis ˜150 nM. The library members are molecules with masses in the 700-750Da range. The solution is infused into a Finnigan LCQ ion trap massspectrometer and ionized by electrospray. A range of 1000-3000 m/z isscanned for ions of the nucleic acid target and its complexes generatedfrom binding with paromomycin and members of the combinatorial library.Typically 200 scans are averaged for 5 minutes.

[0173] The ions from the nucleic acid target (A) are observed at m/z1486.8 for the (M-6H)⁶⁻ ion, 1784.4 for the (M-5H)⁵⁻ ion and 2230.8 forthe (M-4H)⁴⁻ ion. Signals from complexes of target (A) with members ofthe library are expected to occur with m/z values in the 1603.2-1611.6,1924.4-1934.4 and 2405.8-2418.3 ranges.

[0174] Signals from complexes of the nucleic acid target (B), that bearsa mass modifying PEG tag, with members of the combinatorial library areobserved with m/z values in the 2199-2207.4,2639-2649 and 3299-3311ranges. Therefore, the signals of noncovalent complexes with target (B)are cleanly resolved from the signals of complexes arising from thefirst target (A). New signals observed in the mass spectrum aretherefore readily assigned as arising from binding of a library memberto either target (A) or target (B).

[0175] Extension of this mass modifying technique to larger numbers oftargets via the use of unique, high molecular weight neutral andcationic polymers allows for the simultaneous screening of more than twotargets against individual compounds or combinatorial libraries.

Example 8 Simultaneous Screening of a Combinatorial Library of Compoundsagainst Two Peptide Targets

[0176] The two peptide targets to be screened are synthesized usingautomated peptide synthesizers. The first target (A) is a 27-merpolypeptide of known sequence. The second target (B) is also a 27-merpolypeptide that is of identical amino acid composition but completelyscrambled sequence compared to target (A). Target (B) is modified in thelast step of automated synthesis by the addition of a mass modifyingtag, a polyethylene glycol (PEG) chloroformate to its amino terminus.This results in a mass increment of ˜3600 in target (B), which bears amass modifying tag, compared to target (A).

[0177] A solution containing 10 mM target (A) and 10 mM mass modifiedtarget (B) is prepared by dissolving appropriate amounts of both targetsinto 100 mM ammonium acetate at pH 7.4. This solution is treated analiquot of a DMSO solution of the combinatorial library to be screened.The amount of DMSO solution of the library that is added is adjusted sothat the final concentration of each of the 216 member components of thelibrary is ˜150 nM. The library members are molecules with masses in the700-750 Darange. The solution is infused into a Finnigan LCQ ion trapmass spectrometer and ionized by electrospray. A range of 1000-3000 m/zis scanned for ions of the polypeptide target and its complexesgenerated from binding with members of the combinatorial library.Typically 200 scans are averaged for 5 minutes.

[0178] The ions from the polypeptide target (A) and complexes of target(A) with members of the library are expected to occur at much lower m/zvalues that the signals from the polypeptide target (B), that bears amass modifying PEG tag, and its complexes with members of thecombinatorial library Therefore, the signals of noncovalent complexeswith target (B) are cleanly resolved from the signals of complexesarising from the first target (A). New signals observed in the massspectrum are therefore readily assigned as arising from binding of alibrary member to either target (A) or target (B). In this fashion, twoor more peptide targets may be readily screened for binding against anindividual compound or combinatorial library.

Example 9 Gas-phase Dissociation of Nucleic Acids for Determination ofStructure

[0179] Nucleic acid duplexes can be transferred from solution to the gasphase as intact duplexes using electrospray ionization and detectedusing a Fourier transform, ion trap, quadrupole, time-of-flight, ormagnetic sector mass spectrometer. The ions corresponding to a singlecharge state of the duplex can be isolated via resonance ejection,off-resonance excitation or similar methods known to those familiar inthe art of mass spectrometry. Once isolated, these ions can be activatedenergetically via blackbody irradiation, infrared multiphotondissociation, or collisional activation. This activation leads todissociation of glycosidic bonds and the phosphate backbone, producingtwo series of fragment ions, called the w-series (having an intact3′-terminus and a 5′-phosphate following internal cleavage) and thea-Base series (having an intact 5′-terminus and a 3′-furan). Theseproduct ions can be identified by measurement of their mass/charge ratioin an MS/MS experiment.

[0180] An example of the power of this method is presented in FIGS. 8and 9. Shown in FIG. 8 part A is a graphical representation of theabundances of the w and a-Base ions resulting from collisionalactivation of the (M-5H)⁵⁻ ions from a DNA:DNA duplex containing a G-Gmismatch base pair. The w series ions are highlighted in black and pointtoward the duplex, while the a-Base series ions are highlighted in grayand point away from the duplex. The more abundant the fragment ion, thelonger and thicker the respective arrow. Substantial fragmentation isobserved in both strands adjacent to the mismatched base pair. Theresults obtained following collisional activation of the control DNA:DNAduplex ion is shown in FIG. 8 part B. Some product ions are common, butthe pattern of fragmentation differs significantly from the duplexcontaining the mismatched base pair. Analysis of the fragment ions andthe pattern of fragmentation allows the location of the mismatched basepair to be identified unambiguously. In addition, the results suggestthat the gas phase structure of the duplex DNA ion is altered by thepresence of the mismatched pair in a way which facilitates fragmentationfollowing activation.

[0181] A second series of experiments with three DNA:RNA duplexes arepresented in FIG. 9 In the upper figure, an A-C mismatched pair has beenincorporated into the duplex. Extensive fragmentation producing w anda-Base ions is observed adjacent to the mismatched pair. However, theincreased strength of the glycosidic bond in RNA limits thefragmentation of the RNA strand. Hence, the fragmentation is focusedonto the DNA strand. In the central figure, a C-C mismatched base pairhas been incorporated into the duplex, and enhanced fragmentation isobserved at the site of the mismatched pair. As above, fragmentation ofthe RNA strand is reduced relative to the DNA strand. The lower figurecontains the fragmentation observed for the control RNA:DNA duplexcontaining all complementary base pairs. A common fragmentation patternis observed between the G5-T4 bases in all three cases. However, theextent of fragmentation is reduced in the complementary duplexesrelative to the duplexes containing base pair mismatches.

Example 10 MASS Analysis of RNA—Ligand Complex to Determine Binding ofLigand to Molecular Interaction Site

[0182] The ability to discern through mass spectroscopy whether or not aproposed ligand binds to a molecular interaction site of an RNA can beshown. FIGS. 10 and 11 depict the mass spectroscopy of an RNA segmenthaving a stem-loop structure with a ligand, schematically illustrated byan unknown, functionalized molecule. The ligand is combined with the RNAfragment under conditions selected to facilitate binding and the resultin complex is analyzed by a multi target affinity/specificity screening(MASS) protocol. This preferably employs electrospray ionization Fouriertransform ion cyclotron resonance mass spectrometry as describedhereinbefore and in the references cited herein. “Mass chromatography”as described above permits one to focus upon one bimolecular complex andto study the fragmentation of that one complex into characteristic ions.The situs of binding of ligand to RNA can, thus, be determined throughthe assessment of such fragments; the presence of fragmentscorresponding to molecular interaction site and ligand indicating thebinding of that ligand to that molecular interaction site.

[0183]FIG. 10 depicts a MASS Analysis of a Binding Location for a non-ASite Binding molecule. The isolation through “mass chromatography” andsubsequent dissociation of the (M-5H)5− complex is observed at m/z1919.8. The mass shift observed in select fragments relative to thefragmentation observed for the free RNA provides information about wherethe ligand is bound. The (2−) fragments observed below m/z 1200correspond to the stem structure of the RNA; these fragments are notmass shifted upon Complexation. This is consistent with the ligand notbinding to the stem structure.

[0184]FIG. 11 shows a MASS Analysis of Binding Location for the non-ASite Binding molecule. Isolation (i.e. “mass chromatography”) andsubsequent dissociation of the (M-5H)5− complex observed at m/z 1929.4provides significant protection from fragmentation in the vicinity ofthe A-site. This is evidenced by the reduced abundance of the w anda-base fragment ions in the 2300-2500 m/z range. The mass shift observedin select fragments relative to the fragmentation observed for the freeRNA provides information about where the ligand is bound. The exactmolecular mass of the RNA can act as an internal or intrinsic mass labelfor identification of molecules bound to the RNA. The (2-) fragmentsobserved below m/z 1200 correspond to the stem structure of the RNA.These fragments are not mass shifted upon Complexation—consistent withligand not being bound to the stem structure. Accordingly, the locationof binding of ligands to the RNA can be determined.

Example 11 Determination of Specificity and Affinity of Ligand Librariesto RNA Targets

[0185] A preferred first step of MASS screening involves mixing the RNAtarget (or targets) with a combinatorial library of ligands designed tobind to a specific site on the target molecule(s). Specific noncovalentcomplexes formed in solution between the target(s) and any librarymembers are transferred into the gas phase and ionized by ESI. Asdescribed herein, from the measured mass difference between the complexand the free target, the identity of the binding ligand can bedetermined. The dissociation constant of the complex can be determinedin two ways: if a ligand with a known binding affinity for the target isavailable, a relative Kd can be measured by using the known ligand as aninternal control and measuring the abundance of the unknown complex tothe abundance of the control, alternatively, if no internal control isavailable, Kd's can be determined by making a series of measurements atdifferent ligand concentrations and deriving a Kd value from the“titration” curve.

[0186] Because screening preferably employs large numbers of similar,preferably combinatorially derived, compounds, it is preferred that inaddition to determining whether something from the library binds thetarget, it is also determined which compound(s) are the ones which bindto the target. With highly precise mass measurements, the mass identityof an unknown ligand can be constrained to a unique elementalcomposition. This unique mass is referred to as the compound's“intrinsic mass label.” For example, while there are a large number ofelemental compositions which result in a molecular weight ofapproximately 615 Da, there is only one elemental composition(C₂₃H₄₅N₅O₁₄) consistent with a monoisotopic molecular weight of615.2963012 Da. For example, the mass of a ligand (paromomycin in thisexample) which is noncovalently bound to the 16S A-site was determinedto be 615.2969+0.0006 (mass measurement error of 1 ppm) using the freeRNA as an internal mass standard. A mass measurement error of 100 ppmdoes not allow unambiguous compound assignment and is consistent withnearly 400 elemental compositions containing only atoms of C, H, —N, andO. The isotopic distributions shown in the expanded views are primarilya result of the natural incorporation of 13C atoms; because highperformance FTICR can easily resolve the 12C-13C mass difference we canuse each component of the isotopic cluster as an internal mass standard.Additionally, as the theoretical isotope distribution of the free RNAcan be accurately simulated, mass differences can be measured between“homoisotopic” species (in this example the mass difference is measuredbetween species containing four 13C atoms).

[0187] Once the identity of a binding ligand is determined, the complexis isolated in the gas phase (i.e. “mass chromatography”) anddissociated. By comparing the fragmentation patterns of the free targetto that of the target complexed with a ligand, the ligand binding sitecan be determined. Dissociation of the complex is performed either bycollisional activated dissociation (CAD) in which fragmentation iseffected by high energy collisions with neutrals, or infraredmultiphoton dissociation (IRMPD) in which photons from a high power IRlaser cause fragmentation of the complex.

[0188] A 27-mer RNA containing the A-site of the 16S rRNA was chosen asa target for validation experiments. See FIG. 12. The aminoglycosideparomomycin is known to bind to the unpaired adenosine residues with aKd of 200 nM and was used as an internal standard. The target was at aninitial concentration of 10 mM while the paromomycin and each of the 216library members were at an initial concentration of 150 nM. While thisexample was performed on a quadrupole ion trap which does not afford thehigh resolution or mass accuracy of the FTICR, it serves to illustratethe MASS concept. Molecular ions corresponding to the free RNA areobserved at m/z 1784.4 (M-5H+)5− and 2230.8 4 (M-4H+)4−. The signalsfrom the RNA-paromomycin internal control are observed at m/z 1907.1 4(M-5H+)5− and 2384.4 4 (M-4H+)4−. In addition to the expectedparomomycin complex, a number of complexes are observed corresponding tobinding of library members to the target. See FIG. 13.

[0189] One member of this library (MW=675.8+1.5) forms a strong complexwith the target but MS/MS studies reveal that the ligand does not offerprotection of A-site fragmentation and therefore binds to the loopregion. Another member of Isis 113069 having an approximate mass of743.8+1.5 demonstrates strong binding to the target and, as evidenced byMS/MS experiments provides protection of the unpaired A residues,consistent with binding at the A-site.

[0190] The rapid and parallel nature of the MASS approach allows largenumbers of compounds to be screened against multiple targetssimultaneously, resulting in greatly enhanced sample throughput andinformation content. In a single assay requiring less than 15 minutes,MASS can screen 10 targets against a library containing over 500components and report back which compounds bind to which targets, wherethey bind, and with what binding affinity.

Example 12 High Precision ESI-FTICR Mass Measurement of 16S A SiteRNA/Paromomycin Complex

[0191] Electrospray ionization Fourier transform ion cyclotron resonancemass spectrometry was performed on a solution containing 5 mM 16S RNA(the 27-mer construct shown in FIG. 24) and 500 nM paromomycin isdepicted in FIG. 13. A 1:1 complex was observed between the paromomycinand the RNA consistent with specific aminoglycoside binding at theA-site. The insets show the measured and calculated isotope envelopes ofthe (M-5H+)5− species of the free RNA and the RNA-paromomycin complex.High precision mass measurements were acquired using isotope peaks ofthe (M-5H+)⁵⁻ and (M-4H⁺)⁴⁻ charge states of the free RNA as internalmass standards and measuring the m/z difference between the free andbound RNA.

Example 13 Mass of 60-Member Library Against 16S A-Site RNA

[0192] FTMS spectrum was obtained from a mixture of a 16S RNA model (10mM) and a 60-member combinatorial library. Signals from complexes arehighlighted in the insert. Binding of a combinatorial library containing60 members to the 16S RNA model have been examined under conditionswhere each library member was present at 5-fold excess over the RNA. Asshown in FIG. 14, complexes between the 16S RNA and ˜5 ligands in thelibrary were observed.

[0193] An expanded view of the 1863 complex from FIG. 14 is shown inFIG. 15. Two of the compounds in the library had a nominal mass of 398.1Da. Their calculated molecular weights based on molecular formulasindicate that they differ in mass by 46 mDa. Accurate measurement of themolecular mass for the respective monoisotopic (all ¹²C, ¹⁴N, and 16O)[M-5H]⁵− species of the complex (m/z 1863.748) and the free RNA (m/z1784.126) allowed the mass of the ligand to be calculated as398.110±0.009 Da.

[0194]FIG. 16 shows high resolution ESI-FTICR spectrum of the libraryused in FIGS. 14 and 15, demonstrating that both library members with anominal molecular weight of 398.1 were present in the synthesizedlibrary.

Example 14 Compound Identification From a 60-Member CombinatorialLibrary with MASS

[0195] Based on the high precision mass measurement of the complex, themass of the binding ligand was determined to be consistent with thelibrary member having a chemical formula of C₁₅H₁₆N₄O₂F₆ and a molecularweight of 398.117 Da (FIG. 17). Thus, the identity of the binding ligandwas unambiguously established.

Example 15 Elemental Composition Constraints

[0196] Use of exact mass measurements and elemental constraints can beused to determine the elemental composition of an “unknown” bindingligand. General constraints on the type and number of atoms in anunknown molecule, along with a high precision mass measurement, allowdetermination of a limited list of molecular formulas which areconsistent with the measured mass. Referring to FIG. 18, the elementalcomposition is limited to atoms of C, H, N, and O and furtherconstrained by the elemental composition of a “known” moiety of themolecule. Based on these constraints, the enormous number of atomiccombinations which result in a molecular weight of 615.2969±0.0006 arereduced to two possibilities. In addition to unambiguously identifyingintended library members, this technique allows one skilled in the artto identify unintended synthetic by-products which bind to the moleculartarget.

Example 16 Determination Of The MASS K_(d) For 16S-Paromomycin

[0197] The results of direct determination of solution phasedissociation constants (Kd's) by mass spectrometry is shown in FIG. 19.ESI-MS measurements of a solution containing a fixed concentration ofRNA at different concentrations of ligand were obtained. By measuringthe ratio of bound:unbound RNA at varying ligand concentrations, the Kdwas determined by 1/slope of the “titration curve”. The MS derived valueof 110 InM is in good agreement with previously reported literaturevalue of 200 nM.

Example 17 Multi-target Affinity/Specificity Screening

[0198] A schematic representation for the determination of ligandbinding site by tandem mass spectrometry is shown in FIG. 20. A solutioncontaining the molecular target or targets is mixed with a library ofligands and given the opportunity to form noncovalent complexes insolution. These noncovalent complexes are mass analyzed. The noncovalentcomplexes are subsequently dissociated in the gas phase via IRMPD orCAD. A comparison of the fragment ions formed from dissociation of thecomplex with the fragment ions formed from dissociation of the free RNAreveals the ligand binding site.

Example 18 MASS Analysis of 27-Member Library with 16S A-Site RNA

[0199]FIG. 21 shows MASS screening of a 27 member library against a27-mer RNA construct representing the prokaryotic 16S A-site. The insetreveals that a number of compounds formed complexes with the 16S A-site.

Example 19 MASS Protection Assay

[0200] MS/MS of a 27-mer RNA construct representing the prokaryotic 16SA-site containing deoxyadenosine residues at the paromomycin bindingsite is shown in FIG. 22. The top spectrum was acquired by CAD of the[M-5H]5− ion (m/z 1783.6) from uncomplexed RNA and exhibits significantfragmentation at the deoxyadenosine residues. The bottom spectrum wasacquired from by CAD of the [M-5H]5− ion of the 16S-paromomycin complex(m/z 1907.5) under identical activation energy as employed in the topspectrum. No significant fragment ions are observed in the bottomspectrum consistent with protection of the binding site by the ligand.

[0201] Two combinatorial libraries containing 216 tetraazacyclophanesdissolved in DMSO were mixed with a buffered solution containing 10 mM16S RNA (see FIG. 24) such that each library member was present at 100nM. The resulting mass spectra, shown in FIG. 23 reveal >10 complexesbetween 16S RNA and library members with the same nominal mass. MS-MSspectra obtained from a mixture of a 27-mer RNA construct representingthe prokaryotic 16S A-site containing deoxyadenosine residues at theparomomycin binding and the 216 member combinatorial library. In the topspectrum, ions from the most abundant complex from the first library([M-5H];5− m/z 1919.0) were isolated and dissociated. Dissociation ofthis complex generates three fragment ions at m/z 1006.1, 1065.6, and1162.4 that result from cleavage at each dA residue. More intensesignals are observed at m/z 2378.9, 2443.1, and 2483.1. These ionscorrespond to the w21(3−), a20-B(3−), and a21-B(3−) fragments bound to alibrary member with a mass of 676.0±0.6 Da. The relative abundances ofthe fragment ions are similar to the pattern observed for uncomplexedRNA, but the masses of the ions from the lower stem and tetraloop areshifted by complexation with the ligand. This ligand offers littleprotection of the deoxyadenosine residues, and must bind to the lowerstem-loop. The library did not inhibit growth of bacteria. In the bottomspectrum, dissociation of the most abundant complex from a mixture of16S RNA and the second library having m/z 1934.3 with the samecollisional energy yields few fragment ions, the predominant signalsarising from intact complex and loss of neutral adenine. The reducedlevel of cleavage and loss of adenine for this complex is consistentwith binding of the ligand at the model A site region as doesparomomycin. The second library inhibits transcription/translation at 5mM, and has an MIC of 2-20 mM against E. coli(imp-) and S. pyogenes.

Example 20 Neutral Mass Tag of Eukaryotic and Prokaryotic A-Sites

[0202]FIG. 24 shows secondary structures of the 27 base RNA models usedin this work corresponding to the 18S (eukaryotic) and 16S (prokaryotic)A-sites. The base sequences differ in seven positions (bold), the netmass difference between the two constructs is only 15.011 Da. Mass tagswere covalently added to the 5′ terminus of the RNA constructs usingtradition phosphoramadite coupling chemistry.

[0203] Methodology to increase the separation between the associatedsignals in the mass spectra was developed in view of the overlap amongsignals from RNAs 16S and 18S. RNA targets modified with additionaluncharged functional groups conjugated to their 5′-termini weresynthesized. Such a synthetic modification is referred to herein as aneutral mass tag. The shift in mass, and concomitant m/z, of amass-tagged macromolecule moves the family of signals produced by thetagged RNA into a resolved region of the mass spectrum. ESI-FTICRspectrum of a mixture of 27-base representations of the 16S A-site with(7 mM) and without (1 mM) an 18 atom neutral mass tag attached to the5-terminus in the presence of 500 nM paromomycin is shown in FIG. 25.The ratio between unbound RNA and the RNA-paromomycin complex wasequivalent for the 16S and 16S+tag RNA targets demonstrating that theneutral mass tag does not have an appreciable effect on RNA-ligandbinding.

Example 21 Simultaneous Screening of 16S A-Site And 18S A-Site ModelRNAs Against Aminoglycoside Mixture

[0204] Paromomycin, lividomycin (MW=761.354 Da), sisomicin (MW=447.269Da), tobramycin (MW=467.2591 Da), and bekanamycin (MW=483.254 Da) wereobtained from Sigma (St. Louis, Mo.) and ICN (Costa Mesa, Calif.) andwere dissolved to generate 10 mM stock solutions. 2′ methoxy analogs ofRNA constructs representing the prokaryotic (16S) rRNA and eukaryotic(18S) rRNA A-site (FIG. 24) were synthesized in house and precipitatedtwice from 1 M ammonium acetate following deprotection with ammonia (pH8.5). The mass-tagged constructs contained an 18-atom mass tag(C₁₂H₂₅O₉) attached to the 5′-terminus of the RNA oligomer through aphosphodiester linkage.

[0205] All mass spectrometry experiments were performed using an Apex II70e electrospray ionization Fourier transform ion cyclotron resonancemass spectrometer (Bruker Daltonics, Billerica) employing an activelyshielded 7 tesla superconducting magnet. RNA. solutions were prepared in50 mM NH₄OAc (pH 7), mixed 1:1 v:v with isopropanol to aid desolvation,and infused at a rate of 1.5 mL/min using a syringe pump. Ions wereformed in a modified electrospray source (Analytica, Branford) employingan off axis, grounded electrospray probe positioned ca. 1.5 cm from themetalized terminus of the glass desolvation capillary biased at 5000 V.A counter-current flow of dry oxygen gas heated to 225° C. was employedto assist in the desolvation process. Ions were accumulated in anexternal ion reservoir comprised of an RF-only hexapole, a skimmer cone,and an auxiliary electrode for 1000 ms prior to transfer into thetrapped ion cell for mass analysis. Each spectrum was the result of thecoaddition of 16 transients comprised of 256 datapoints acquired over a90,909 kHz bandwidth resulting in a 700 ms detection interval. Allaspects of pulse sequence control, data acquisition, and postacquisition processing were performed using a Bruker Daltonicsdatastation running XMASS version 4.0 on a Silicon Graphics (San Jose,Calif.) R5000 computer.

[0206] Mass spectrometry experiments were performed in order to detectcomplex formation between a library containing five aminoglycosides(Sisomicin (Sis), Tobramycin (Tob), Bekanomycin (Bek), Paromomycin (PM),and Livodomycin (LV)) and two RNA targets simultaneously. Signals fromthe (M-5H+)⁵− charge states of free 16S and 18S RNAs are detected at m/z1801.515 and 1868.338, respectively. As shown in FIG. 26, the massspectrometric assay reproduces the known solution binding properties ofaminoglycosides to the 16S A site model and an 18S A site model with aneutral mass linker. Consistent with the higher binding affinity oftheses aminoglycosides for the 16S A-site relative to the 18S A-site,aminoglycoside complexes are observed only with the 16S rRNA target.Note the absence of l8S-paromomycin and 18S-lividomycin complexes, whichwould be observed at the m/z's indicated by the arrows. The insetdemonstrates the isotopic resolution of the complexes. Using multipleisotope peaks of the (M-5H+)⁵⁻ and (M-4H+)⁴⁻ charge states of the freeRNA as internal mass standards, the average mass measurement error ofthe complexes is 2.1 ppm. High affinity complexes were detected betweenthe 16S A site 27mer RNA and paromomycin and lividomycin, respectively.Weaker complexes were observed with sisomycin, tobramycin and bekamycin.No complexes were observed between any of the aminoglycosides and the18S A site model. Thus, this result validates the mass spectrometricassay for identifying compounds that will bind specifically to thetarget RNAs. No other type of high throughput assay can provideinformation on the specificity of binding for a compound to two RNAtargets simultaneously. The binding of lividomycin to the 16S A site hadbeen inferred from previous biochemical experiments. The massspectrometer has been used herein to measure a K_(D) of 28 nM forlividomycin and 110 nM for paromomycin to the 16S A site 27mer. Thesolution K_(D) for paromomycin has been estimated to be between 180 nMand 300 nM.

Example 22 Targeted Site-specific Gas-phase Cleavage ofOligoribonucleotides—Application in Mass Spectrometry-basedIdentification of Ligand Binding Sites

[0207] Fragmentation of oligonucleotides is a complex process, butappears related to the relative strengths of the glycosidic bonds. Thisobservation is exploited by incorporating deoxy-nucleotides selectivelyinto a chimeric 2′-O-methylribonucleotide model of the bacterial rRNA Asite region. Miyaguchi, et al., Nucl. Acids Res., 1996, 24, 3700-3706;Fourmy, et al., Science, 1996,274, 1367-1371; and Fourmy, et al., J Mol.Biol., 1998,277, 333-345. During CAD, fragmentation is directed to themore labile deoxynucleotide sites. The resulting CAD mass spectrumcontains a small subset of readily assigned complementary fragment ions.Binding of ligands near the deoxyadenosine residues inhibits the CADprocess, while complexation at remote sites does not affect dissociationand merely shifts the masses of specific fragment ions. These methodsare used to identify compounds from a combinatorial library thatpreferentially bind to the RNA model of the A site region.

[0208] The 27-mer model of a segment of the bacterial A site region hasbeen prepared as a full ribonucleotide (see FIG. 27, compound R), and asa chimeric 2′-O-methylribonucleotide containing three deoxyadenosineresidues (see FIG. 27, compound C). RNAs R and C have been preparedusing conventional phosphoramidite chemistry on solid support.Phosphoramidites were purchased from Glen Research and used as 0.1 Msolutions in acetonitrile. RNA R was prepared following the proceduregiven in Wincott, et al., Nucl. Acids Res., 1995, 23, 2677-2684, thedisclosure of which is incorporated herein by reference in its entirety.RNA C was prepared using standard coupling cycles, deprotected, andprecipitated from 10 M NH₄OAc. The aminoglycoside paromomycin binds toboth R and C with kD values of 0.25 and 0.45 micromolar, respectively.The reported kD values are around 0.2 μM. Recht, et al., J. Mol. Biol.,1996, 262, 421-436, Wong, et al., Chem. Biol., 1998, 5, 397-406, andWang, et al., Biochemistry, 1997, 36, 768-779. Paromomycin has beenshown previously to bind in the major groove of the 27mer model RNA andinduce a conformational change, with contacts to A1408, G1494, andG1491. Miyaguchi, et al., Nucl. Acids Res., 1996, 24, 3700-3706; Fourmy,et al., Science, 1996, 274, 1367-1371; and Fourmy, et al, J. Mol. Biol.,1998, 277, 333-345.

[0209] The mass spectrum obtained from a 5 μM solution of C mixed with125 nM paromomycin (FIG. 28A) contains [M-5H]5− ions from free C at m/z1783.6 and the [M-5H]5− ions of the paromomycin-C complex at m/z 1907.3.Mass spectrometry experiments have been performed on an LCQ quadrupoleion trap mass spectrometer (Finnigan; San Jose, Calif.) operating in thenegative ionization mode. RNA and ligand were dissolved in a 150 mMammonium acetate buffer at pH 7.0 with isopropyl alcohol added (1:1 v:v)to assist the desolvation process. Parent ions have been isolated with a1.5 m/z window, and the AC voltage applied to the end caps was increaseduntil about 70% of the parent ion dissociates. The electrospray needlevoltage was adjusted to −3.5 kV, and spray was stabilized with a gaspressure of 50 psi (60:40 N2:02). The capillary interface was heated toa temperature of 180° C. The He gas pressure in the ion trap was 1mTorr. In MS-MS experiments, ions within a 1.5 Da window having thedesired m/z were selected via resonance ejection and stored with q) 0.2.The excitation RF voltage was applied to the end caps for 30 ms andincreased manually to 1.1 Vpp to minimize the intensity of the parention and to generate the highest abundance of fragment ions. A total of128 scans were summed over m/z 700-2700 following trapping for 100 ms.Signals from the [M-4H]4− ions of C and the complex are detected at m/z2229.8 and 2384.4, respectively. No signals are observed from morehighly charged ions as observed for samples denatured withtripropylamine. In analogy with studies of native and denaturedproteins, this is consistent with a more compact structure for C and theparomomycin complex. The CAD mass spectrum obtained from the [M-5H]5−ion of C is presented in FIG. 28B. Fragment ions are detected at m/z1005.6 (w6)2−, 1065.8 (a7-B)2−, 1162.6 (w7)2−, 1756.5 (M-Ad)5−, 2108.9(w21-Ad)3−, 2153.4 (a20-B)3−, 2217.8 (w21)3−, and 2258.3 (a21-B)3−.McLuckey, et al., J. Am. Soc. Mass Spectrum., 1992, 3,60-70 andMcLuckey, et al., J. Am. Chem. Soc., 1993, 115, 12085-12095. Thesefragment ions all result from loss of adenine from the threedeoxyadenosine nucleotides, followed by cleavage of the 3′-C—O sugarbonds. The CAD mass spectrum for the [M-5H]5− ion of the complex betweenC and paromomycin obtained with the same activation energy is shown inFIG. 28C. No fragment ions are detected from strand cleavage at thedeoxyadenosine sites using identical dissociation conditions of FIG.28B. The change in fragmentation pattern observed upon binding ofparomomycin is consistent with a change in the local chargedistribution, conformation, or mobility of A1492, A1493, and A1408 thatprecludes collisional activation and dissociation of the nucleotide.

[0210] Two combinatorial libraries containing 216 tetraazacyclophanesdissolved in DMSO were mixed with a buffered solution containing 10 μM Csuch that each library member is present at 100 nM. The resulting massspectra reveal >10 complexes between C and library members with the samenominal mass. Ions from the most abundant complex from the first library([M-5H];⁵⁻ m/z 1919.0) were isolated and dissociated. As shown in FIG.29A, dissociation of this complex generates three fragment ions at m/z1006.1, 1065.6, and 1162.4 that result from cleavage at each dA residue.More intense signals are observed at m/z 2378.9, 2443,1, and 2483.1.These ions correspond to the w₂₁ ⁽³⁻⁾, a₂₀-B⁽³⁻⁾, and a₂₁-B⁽³⁻⁾fragments bound to a library member with a mass of 676.0=0.6Da. Therelative abundances of the fragment ions are similar to the patternobserved for uncomplexed C, but the masses of the ions from the lowerstem and tetraloop are shifted by complexation with the ligand. Thisligand offers little protection of the deoxyadenosine residues, and mustbind to the lower stem-loop. The libraries have been synthesized from amixture of charged and aromatic functional groups, and are described aslibraries 25 and 23 in: An, et al., Bioorg. Med. Chem. Lett., 1998, inpress. Dissociation of the most abundant complex from a mixture of C andthe second library having m/z 1934.3 with the same collisional energy(FIG. 29B) yields few fragment ions, the predominant signals arisingfrom intact complex and loss of neutral adenine. The mass of the ligand(753.5 Da) is consistent with six possible compounds in the libraryhaving two combinations of functional groups. The reduced level ofcleavage and loss of adenine from this complex is consistent withbinding of the ligand at the model A site region as does paromomycin.The second library inhibits transcription/translation at 5 μm, and hasan MIC of 2-20 μM against E. coli (imp-) and S. pyogenes.

[0211] Mass spectrometry-based assays provide many advantages foridentification of complexes between RNA and small molecules. Allconstituents in the assay mixture carry an intrinsic mass label, and noadditional modifications with radioactive or fluorescent tags arerequired to detect the formation of complexes. The chemical compositionof the ligand can be ascertained from the measured molecular mass of thecomplex, allowing rapid deconvolution of libraries to identify leadsagainst an RNA target. Incorporation of deoxynucleotides into a chimericoligoribonicleotide generates a series of labile sites wherecollisionally-activated dissociation is favored. Binding of ligands atthe labile sites affords protection from CAD observed in MS-MSexperiments. This mass spectrometry-based protection methods of theinvention can be used to establish the binding sites for small moleculeligands without the need for additional chemical reagents orradiobabeling of the RNA. The methodology can also be used in DNAsequencing and identification of genomic defects.

[0212] In accordance with preferred embodiments of the presentinvention, enhanced accuracy of determination of binding between targetbiomolecules and putative ligands is desired. It has been found thatcertain mass spectrometric techniques can give rise to such enhancement.As will be appreciated, the target biomolecule will always be present inexcess in samples to be spectroscopically analyzed. The exactcomposition of such target will, similarly, be known. Accordingly, theisotopic abundances ofthe parent (and other) ions deriving from thetarget will be known to precision.

[0213] In accordance with preferred embodiments, mass spectrometric datais collected from a sample comprising target biomolecule (orbiomolecules) which has been contacted with one or more, preferably amixture of putative or trial ligands. Such a mixture of compounds may bequite complex as discussed elsewhere herein. The resulting mass spectrumwill be complex as well, however, the signals representative of thetarget biomolecule(s) will be easily identified. It is preferred thatthe isotopic peaks for the target molecule be identified and used tointernally calibrate the mass spectrometric data thus collected sincethe M/e for such peaks is known with precision. As a result, it becomespossible to determine the exact mass shift (with respect to the targetsignal) of peaks which represent complexes between the target andligands bound to it. Given the exact mass shifts, the exact molecularweights of said ligands may be determined. It is preferred that theexact molecular weights (usually to several decimal points of accuracy)be used to determine the identity of the ligands which have actuallybound to the target.

[0214] In accordance with other preferred embodiments, the informationcollected can be placed into a relational or other database, from whichfurther information concerning ligand binding to the target biomoleculecan be extracted. This is especially true when the binding affinities ofthe compounds found to bind to the target are determined and included inthe database. Compounds having relatively high binding affinities can beselected based upon such information contained in the database.

[0215] It is preferred that such data collection and databasemanipulation be achieved through a general purpose digital computer. Anexemplary software program has been created and used to identify thesmall molecules bound to an RNA target, calculate the binding constant,and write the results to a relational database. The program uses asinput a file that lists the elemental formulas of the RNA and the smallmolecules which are present in the mixture under study, and theirconcentrations in the solution. The program first calculates theexpected isotopic peak distribution for the most abundant charge stateof each possible complex, then opens the raw FTMS results file. Theprogram performs a fast Fourier transform of the raw data, calibratesthe mass axis, and integrates the signals in the resulting spectrum suchas the exemplary spectrum shown in FIG. 30. The peaks in the spectrumare preferably identified via centroiding as shown in FIG. 31, areintegrated, and preferably stored in a database. An exemplary data fileis shown in FIG. 32). The expected and observed peaks are correlated,and the integrals converted into binding constants based on theintensity of an internal standard. The compound identity and bindingconstant data are written to a relational database. This approach allowslarge amounts of data that are generated by the mass spectrometer to beanalyzed without human intervention, which results in a significantsavings in time.

[0216]FIG. 30 depicts electrospray ionization Fourier transform ioncyclotron resonance mass spectrometry of a solution which is 5 mM in 16SRNA (Ibis 16628) and 500 nM in the ligand Ibis 10019. The rawtime-domain dataset is automatically apodized and zerofilled twice priorto Fourier transformation. The spectrum is automatically post-calibratedusing multiple isotope peaks of the (M-5H⁺)⁵⁻ and (M-4H⁺)⁴⁻ chargestates of the free RNA as internal mass standards and measuring the m/zdifference between the free and bound RNA. The isotope distribution ofthe free RNA is calculated a priori and the measured distribution is fitto the calculated distribution to ensure that m/z differences aremeasured between homoisotopic species (e.g. monoisotopic peaks orisotope peaks containing 4 ¹³C atoms).

[0217]FIG. 31 shows isotope clusters observed in the m/z range whereRNA-ligand complexes are expected are further analyzed by peakcentroiding and integration. FIG. 32 depicts data tabulated and storedin a relational database. Peaks which correspond to complexes betweenthe RNA target and ligands are assigned and recorded in the database. Ifan internal affinity standard is employed, a relative Kd isautomatically calculated from the relative abundance of the standardcomplex and the unknown complex and recorded in the database. FIG. 33depicts a flow chart for one computer program for effectuating certainaspects of the present invention.

[0218] When computer controlled collection of the foregoing informationis provided and computer control of relational databases is employed,the present invention is capable of very high throughput analysis ofmass spectrometric binding information. Such control facilitates theidentification of ligands having high binding affinities for the targetbiomolecules. Thus, automation permits the automatic calculation of themass of the binding ligand or ligands, especially when the mass of thetarget is used for internal calibration purposes. From the precise massof the binding ligands, their identity may be determined in an automatedway. The dissociation constant for the ligand—target interaction mayalso be ascertained using either known Kd and abundance of a referencecomplex or by titration with multiple measurements at differenttarget/ligand ratios. Further, tandem mass spectrometric analyses may beperformed in an automated fashion such that the site of the smallmolecule, ligand, interaction with the target can be ascertained throughfragmentation analysis. Computer input and output from the relationaldatabase is, of course, preferred.

1 2 1 27 RNA Artificial Sequence Description of Artificial SequenceOLIGONUCLEOTIDE 1 ggcgucacac cuucggguga agucgcc 27 2 27 RNA ArtificialSequence Description of Artificial Sequence OLIGONUCLEOTIDE 2 ggcgucacaccuucggguga agucgcc 27

What is claimed is:
 1. A method for determining three dimensionalstructure of a nucleic acid comprising: (a) providing a chimeric versionof said nucleic acid having at least one modified subunit in apreselected position of the chimera; (b) ionizing said nucleic acidhybrid in a mass spectrometer to provide one or more ions of saidchimera; (c) fragmenting at least one of said ions; (d) collectingfragmentation data from said fragmentation; and (e) relating saidfragmentation data to said three dimensional structure.
 2. The method ofclaim 1 wherein said relating of fragmentation data comprisesidentification of the fragmentation pattern of said ions.
 3. The methodof claim 1 wherein said three-dimensional structure comprises asecondary or tertiary structures.
 4. The method of claim 1 wherein saidthree dimensional structure comprises at least one mismatched base pair,loop, bulge, kink or stem structure.
 5. The method of claim 1 whereinsaid nucleic acid is RNA.
 6. The method of claim 5 wherein said nucleicacid corresponds to a 16S rRNA A-site.
 7. The method of claim 1 whereinsaid nucleic acid chimera comprises RNA having one or moredeoxynucleotides at said preselected positions.
 8. The method of claim 1wherein said chimera comprises RNA or DNA having a nucleic acid analogmoieties at said preselected positions.
 9. The method of claim 1 whereinsaid ionizing of said chimera is achieved through electrosprayionization, atmospheric pressure ionization or matrix-assisted laserdesorption ionization.
 10. The method of claim 1 wherein said ionfragmentation takes place within a mass spectrometer capable ofperforming quadrupole, triple quadrupole, magnetic sector, ion trap, ioncyclotron resonance or time-of-flight mass spectrometry.
 11. The methodof claim 1 wherein said ion fragmentation involves collision-induceddissociation.
 12. The method of claim 1 wherein said ion fragmentationinvolves infrared multiphoton dissociation.
 13. A method for identifyinga binding site for a ligand on a biomolecular target comprising: (a)collecting mass spectral fragmentation data for said biomoleculartarget; (b) providing a complex of said biomolecular target and saidbinding agent; (c) ionizing said complex in a mass spectrometer toprovide one or more ions of said complex; (d) fragmenting at least oneof said ions deriving from the complex; (e) collecting fragmentationdata from said fragmentation of the ions from the complex; and (f)relating the fragmentation data of the ions of the complex with thefragmentation data from the biomolecular target to determine said siteof binding.
 14. The method of claim 13 wherein said biomolecular targetis a nucleic acid.
 15. The method of claim 13 wherein said biomoleculartarget is RNA.
 16. The method of claim 15 wherein said RNA correspondsto a 16S rRNA A-site.
 17. The method of claim 14 wherein said nucleicacid includes at least one non-native nucleotide or nucleotide analog atpreselected positions thereof.
 18. The method of claim 17 comprising DNAat said preselected positions of an RNA.
 19. The method of claim 13wherein said biomolecular target is a peptide, protein, antibody,carbohydrate, oligosaccharide or glycopeptide.
 20. The method of claim13 wherein said biomolecular target is a nucleic acid moiety.
 21. Themethod of claim 13 wherein said mass spectral fragmentation data forsaid biomolecular target is provided by: (i) ionizing said biomoleculartarget in a mass spectrometer to provide one or more ions of saidbiomolecular target; and (ii) fragmenting in a mass spectrometer atleast one of said ions.
 22. The method of claim 13 wherein said ionizingof said complex is achieved through electrospray ionization, atmosphericpressure ionization or matrix-assisted laser desorption ionization. 23.The method of claim 13 wherein said ion fragmentation takes place withina mass spectrometer capable of performing quadrupole, triple quadrupole,magnetic sector, ion trap, ion cyclotron resonance or time-of-flightmass spectrometry.
 24. The method of claim 13 wherein said ionfragmentation involves collision-induced dissociation.
 25. The method ofclaim 13 wherein said ion fragmentation involves infrared multiphotondissociation.
 26. The method of claim 13 wherein said complex isprovided by combining together said biomolecular target and said bindingagent.
 27. A method for determining the relative binding affinity of abinding agent for a biomolecular target comprising: (a) providing afirst complex of said biomolecular target and said binding agent; (b)ionizing said first complex in a mass spectrometer to provide one ormore ions of said first complex; (c) collecting mass spectral data fromthe ionization of step (b) and identifying therefrom the ion abundanceof said first complex; (d) providing a second complex of saidbiomolecular target and a standard binding compound which binds to saidtarget; (e) ionizing said second complex in a mass spectrometer toprovide one or more ions of said second complex; (f) collecting massspectral data from the ionization of step (e) and identifying therefromthe ion abundance of said second complex, wherein the relative the ionabundances of said first and second complexes affords a measure of saidrelative binding affinity.
 28. The method of claim 27 wherein said ionabundances of said first and second complexes are compared to identifysaid relative binding affinity.
 29. The method of claim 27 wherein saidbiomolecular target is a nucleic acid.
 30. The method of claim 29wherein said nucleic acid is RNA.
 31. The method of claim 30 whereinsaid RNA corresponds to a 16S rRNA A-site.
 32. The method of claim 30wherein said RNA includes one or more deoxynucleotide subunits atpreselected locations thereof.
 33. The method of claim 27 wherein saidbiomolecular target is a peptide, protein, antibody, carbohydrate,oligosaccharide or glycopeptide.
 34. The method of claim 27 wherein saidbiomolecular target is a nucleic acid moiety.
 35. The method of claim 27wherein said ionizing is achieved through electrospray ionization,atmospheric pressure ionization or matrix-assisted laser desorptionionization.
 36. A method for identifying a compound which binds to apreselected biomolecular target, said compound being present in amixture of compounds comprising: (a) providing a complex of saidbiomolecular target and a standard binding compound which binds to saidtarget under conditions effective to achieve said binding; (b) combiningwith said complex under competitive binding conditions the mixture ofcompounds; (c) ionizing said combination in a mass spectrometer toprovide a plurality of ions; (d) fragmenting at least one of said ionsin a mass spectrometer; (e) collecting mass spectral data for thefragmentation; and (f) relating said mass spectral data to the existenceand degree of competitive binding.
 37. The method of claim 36 whereinsaid biomolecular target is a nucleic acid.
 38. The method of claim 37wherein said nucleic acid is RNA.
 39. The method of claim 38 whereinsaid RNA includes one or more deoxynucleotides at preselected locationsthereof.
 40. The method of claim 36 wherein said biomolecular target isa peptide, protein, antibody, carbohydrate, oligosaccharide orglycopeptide.
 41. A method for identifying a compound which binds to apreselected biomolecular target, said compound being present in amixture of compounds comprising: (a) providing a complex of saidbiomolecular target and a standard compound which binds to said targetunder conditions effective to achieve said binding; (b) acquiringfragmentation data from the mass spectrometric analysis of the complex;(c) combining with a further portion of said complex under competitivebinding conditions the mixture of compounds; (d) ionizing saidcombination in a mass spectrometer to provide a plurality of ions; (e)fragmenting at least one of said ions in a mass spectrometer; (f)collecting mass spectral data for the fragmentation; and (g) relatingthe mass spectral data acquired in steps (b) and (f) to the existenceand degree of competitive binding of said compound.
 42. A method foridentifying in a combinatorial mixture compounds which bind to abiomolecular target, wherein the method comprises: (a) providing massspectral data on the ion abundance for said biomolecular target; (b)providing a first complex of said biomolecular target and a standardbinding compound which binds to said target; (c) combining with saidfirst complex a combinatorial mixture of compounds; (d) ionizing in amass spectrometer said combination from step {circle over (c)} toprovide a plurality of ions for said combination; (e) collecting fromthe ionization of step (d) mass spectral data on the ion abundance ofsaid first complex, wherein said ion abundances in steps (a) and (e)affords information for effecting said determination.
 43. The method ofclaim 42 wherein mass differences in the mass spectral data from steps(a) and (e) are identified for determining the mass of compounds fromthe combinatorial mixture which preferentially bind with saidbimolecular target.
 44. The method of claim 42 wherein said biomoleculartarget is a nucleic acid.
 45. The method of claim 44 wherein saidnucleic acid is RNA.
 46. The method of claim 45 wherein said RNAcorresponds to a 16S rRNA A-site.
 47. The method of claim 45 whereinsaid RNA includes one or more deoxynucleotide subunits at preselectedlocations thereof.
 48. The method of claim 47 wherein said biomoleculartarget is a peptide, protein, antibody, carbohydrate, oligosaccharide orglycopeptide.
 49. A method for identifying binding sites ofabiomolecular target for compounds from a combinatorial librarycomprising: (a) providing mass spectral fragmentation data for saidbiomolecular target; (b) providing a first complex of said biomoleculartarget and a standard binding compound which binds to said target; (c)combining with said first complex a combinatorial mixture of compounds;(d) ionizing in a mass spectrometer said combination from step {circleover (c)} to provide a plurality of ions for said combination; (e)fragmenting at least one of said ions in a mass spectrometer to generatefragmentation data; (f) relating the fragmentation data collected forsaid biomolecular target and the ionized combination from step (d) toafford said identification.
 50. The method of claim 49 wherein said massspectral data for said biomolecular target and said combination arecompared to identify said binding sites.
 51. The method of claim 49wherein said biomolecular target is a nucleic acid.
 52. The method ofclaim 51 wherein said nucleic acid is RNA.
 53. A method for determiningthe relative binding affinity of compounds in a combinatorial mixturefor a biomolecular target comprising: (a) providing a first complex ofsaid biomolecular target and a standard binding compound which binds tosaid target; (b) combining with said first complex a combinatorialmixture of compounds, wherein one or more of said compounds from saidcombinatorial mixture preferentially binds with said biomolecular targetto provide secondary complexes; (c) ionizing said combination of step(b) in a mass spectrometer to provide a. plurality of ions; (d)collecting mass spectral data from the ionization of step {circle over(c)} and identifying therein ion abundances for said first and saidsecondary complexes, wherein the ion abundances of said first andadditional complexes affords information for identifying said. relativebinding affinity.
 54. The method of claim 53 wherein said ion abundancesof said first and additional complexes are compared to identify saidrelative binding affinity.
 55. The method of claim 53 wherein saidbiomolecular target is a nucleic acid.
 56. A method for screening aplurality of biomolecular targets against a binding agent comprising:(a) providing in different biomolecular targets, wherein n is an integergreater than or equal to 2; (b) modifying n−1 of said biomoleculartargets with mass modifying tags, wherein the mass to charge ratio ofions of about the same charge of said modified biomolecular targets aresubstantially distinguishable by mass spectrometry; (c) combining saidmodified biomolecular targets with a binding agent; (d) ionizing saidcombination from step {circle over (c)} in a mass spectrometer toprovide a plurality of ions; and (e) collecting mass spectral data fromthe ionization of step (d) and identifying therefrom ion abundances ofsaid modified biomolecular targets and any complexes formed between saidbinding agent and said modified biomolecular targets, wherein said ionabundances afford information for effecting said screening.
 57. Themethod of claim 56 wherein the ion abundances of said ions of saidmodified biomolecular target and said complexes are compared todetermine the selectivity of the binding interaction between said ligandand said targets.
 58. The method of claim 56 wherein said biomoleculartarget is a nucleic acid.
 59. The method of claim 56 wherein said massmodifying tags are polymeric
 60. The method of claim 59 wherein saidpolymers are a polyethylene glycol, polypropylene, polystyrene,cellulose, sephadex, dextran, peptide or polyacrylamide.
 61. The methodof claim 59 wherein said mass modifying tag is attached to the3′-terminus, 5′-terminus or sugar-phosphate backbone of saidbiomolecular targets.
 62. A method for screening a plurality ofbiomolecular targets against a combinatorial library of compoundscomprising: (a) providing n different biomolecular targets, wherein n isan integer greater than or equal to 2; (b) modifying n−1 of saidbiomolecular targets with mass modifying tags, wherein the mass tocharge ratio of ions of about the same charge of said modifiedbiomolecular targets are substantially distinguishable by massspectrometry; (c) combining said modified biomolecular targets with saidcombinatorial library of compounds; (d) ionizing said combination fromstep {circle over (c)} in a mass spectrometer to provide a plurality ofions; and (e) collecting mass spectral data from the ionization of step(d) and identifying therefrom ion abundances of said modifiedbiomolecular targets and any complexes formed between said compounds andsaid modified biomolecular targets, wherein said ion abundances affordinformation for effecting said screening.
 63. The method of claim 62wherein the ion abundances of said ions of said modified biomoleculartarget and said complexes are compared to determine the selectivity ofthe binding interactions between said compounds and said targets. 64.The method of claim 62 wherein said biomolecular target is a nucleicacid.
 65. The method of claim 62 wherein said mass modifying tags arepolymeric.
 66. The method of claim 62 wherein said mass modifying tag isattached to the 3′-terminus, 5′-terminus or sugar-phosphate backbone ofsaid biomolecular targets.
 67. A method of screening multiplebiomolecular targets against a ligand comprising: (a) providing at leasttwo biomolecular targets which possess different masses such that themass to charge ratio of ions of about the same charge of saidbiomolecular targets are substantially distinguishable by massspectrometry; (b) combining said bimolecular targets with said ligand;(c) ionizing said combination of step (b) in a mass spectrometer to forma plurality of ions; and (d) collecting mass spectral data from theionization of step {circle over (c)} and identifying therefrom ionabundances of said biomolecular targets and any complexes formed betweensaid ligand and said biomolecular targets, wherein said ion abundancesafford information for effecting said screening.
 68. The method of claim67 wherein said ion abundances of said biomolecular target and saidcomplexes are compared to determine the selectivity of the bindinginteractions between said bimolecular targets and said ligand.
 69. Themethod of claim 67 wherein said biomolecular target is a nucleic acid.70. The method of claim 67 wherein said nucleic acid is derived fromprokaryotic or eukaryotic nucleic acids.
 71. The method of claim 67wherein said biomolecular targets comprise a mixture of proteins.
 72. Amethod for determining the nature and extent of binding of a ligand witha molecular interaction site of a biomolecule comprising contacting thebiomolecule with the ligand under conditions selected to promote bindingof the ligand to the molecular interaction site to give rise to acomplex of said biomolecule and the ligand; ionizing the complex in amass spectrometer; fragmenting the ionized complex; and determiningwhether the ligand binds to the molecular interaction site and, if so,determining the strength of binding of such ligand as compared to otherligands bound to said molecular interaction site.
 73. The method ofclaim 72 wherein said biomolecule is RNA.
 74. The method of claim 72wherein said ionization is electrospray ionization.
 75. The method ofclaim 72 wherein said ligand is part of a library of compounds and saidbinding takes place in the presence of other members of the library. 76.The method of claim 72 wherein said fragmentation comprises collisionalactivated dissociation or infrared multiphoton dissociation.
 77. Themethod of claim 72 wherein said determining comprises Fourier transformion cyclotron resonance mass spectroscopy.
 78. A method of identifyingchemical ligands which bind with high specificity and affinity to amolecular interaction site of an RNA comprising preparing a library ofligands in accordance with a ranked hierarchy of such ligands predictedor calculated to be bindable to the molecular interaction site;contacting the RNA with the ligand library under conditions selected topromote binding of the ligand library to the molecular interaction siteof the RNA to give rise to complexes of said RNA and the ligands of thelibrary; ionizing the complexes in a mass spectrometer; fragmenting theionized complexes; and determining whether the ligand of each suchcomplex binds to the molecular interaction site of the RNA and, if so,determining the strength of binding of such ligand as compared to thebinding strength of other ligands of the library.
 79. The method ofclaim 78 wherein said ionization is electrospray ionization.
 80. Themethod of claim 78 wherein said fragmentation comprises collisionalactivated dissociation or infrared multiphoton dissociation.
 81. Themethod of claim 78 wherein said determining comprises Fourier transformion cyclotron resonance mass spectroscopy.
 82. The method of claim 13wherein said binding site is a metal ion binding site.
 83. The method ofclaim 82 wherein said metal ion is an alkali metal or alkaline earthmetal.
 84. The method of claim 83 wherein said metal ion is selectedfrom the group consisting of Na⁺, Mg⁺⁺, and Mn⁺⁺.
 85. The method ofclaim 49 wherein said binding site is a metal ion binding site.
 86. Themethod of claim 85 wherein said metal ion is an alkali metal or alkalineearth metal.
 87. The method of claim 86 wherein said metal ion isselected from the group consisting of Na⁺, Mg⁺⁺, and Mn⁺⁺.
 88. Themethod of claim 13 further comprising determining the absolute bindingaffinity of at least one binding agent and biomolecular target.
 89. Themethod of claim 49 further comprising determining the absolute bindingaffinity of at least one binding agent and biomolecular target.
 90. Amethod for identifying in a chemical mixture of compounds which bind toa biomolecular target, comprising: (a) providing mass spectral data onthe ion abundances for a blend of the mixture and an excess of saidtarget; (b) calibrating the mass spectral data by reference to themultiple isotope peaks for the uncomplexed target; (c) determining theexact mass shift of mass spectral data representing a subset of saidcompounds complexed with said target to ascertain the exact molecularweight of said complexed compounds.
 91. The method of claim 90 furthercomprising: (d) identifying the compounds from among those comprisingthe chemical mixture by reference to their exact molecular weights. 92.The method of claim 91 further comprising establishing a relationaldatabase to hold information collected in said determining andidentifying steps.
 93. The method of claim 92 further comprisingdetermining the binding affinity of the compounds found to bind to saidtarget and including said binding affinity data in said database. 94.The method of claim 93 further comprising selecting from among saidcompounds found to bind to said target, those having relatively highbinding affinity.